METHODS OF USING ANTI-CD79b IMMUNOCONJUGATES

ABSTRACT

Provided herein are methods of treating B-cell proliferative disorders in particular Follicular Lymphoma and/or Diffuse Large B-Cell Lymphoma using immunoconjugates comprising anti-CD79b antibodies in combination with additional therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/358,410, filed Mar. 19, 2019, now issued as U.S. Pat. No. 11,000,510,which is a continuation of U.S. application Ser. No. 15/440,917, filedFeb. 23, 2017, now abandoned, which is a continuation of U.S.application Ser. No. 14/863,125, filed Sep. 23, 2015, now abandoned,which claims the benefit of U.S. Provisional Application Ser. No.62/136,324, filed Mar. 20, 2015, U.S. Provisional Application Ser. No.62/076,823, filed Nov. 7, 2014, and U.S. Provisional Application Ser.No. 62/054,257, filed Sep. 23, 2014, the contents of each of which areincorporated herein by reference in their entireties.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392046010SEQLIST.TXT,date recorded: Jun. 23, 2020, size: 64 KB).

FIELD OF THE INVENTION

Provided herein are methods of treating B-cell proliferative disordersin particular Follicular Lymphoma and/or Diffuse Large B-Cell Lymphomausing immunoconjugates comprising anti-CD79b antibodies in combinationwith additional therapeutic agents.

BACKGROUND OF THE INVENTION

CD79b is the signaling component of the B-cell receptor which acts as acovalent heterodimer containing CD79a (i.e., Igα or mb-1) and CD79b(i.e., Igβ or B29). CD79b contains an extracellular immunoglobulin (Ig)domain, a transmembrane domain, and an intracellular signaling domain,an immunoreceptor tyrosine-based activation motif (ITAM) domain. CD79 isexpressed on B-cells and, for example, in Non-Hodgkin's Lymphoma cells(NHLs) (Cabezudo et al., Haematologica 84:413-418 (1999); D'Arena etal., Am. J. Hematol. 64: 275-281 (2000); Olejniczak et al., Immunol.Invest. 35: 93-114 (2006)). CD79a and CD79b and sIg are all required forsurface expression of the CD79 (Matsuuchi et al., Curr. Opin. Immunol.13(3): 270-7)).

B-cell proliferative disorders are generally treated with somecombination of surgery, radiation therapy and/or drug treatment.Accumulated empirical clinical experience, supported by animal models,supports the hypothesis that cytotoxic drugs may be more effective whengiven in combination to achieve additive or synergistic effects.However, a caveat to the hypothesis is that success requires the abilityto combine drugs at their respective effective doses withoutunacceptable side-effects and avoiding possible pharmacokineticinteractions. Further, although it may seem reasonable to combine atargeted agent with the standard of care, clinical experience indicatesthat differences in administration regimens and the dosages of eachagents has an effect on efficacy of the treatment. These factors haveled to the clinical failure of many combinations. See, e.g., Al-Lazikaniet al., Nature Biotechnology 30:679-692 (2012). There is a need in theart for new treatment regimens for treating B-cell proliferativedisorders including treatments comprising agents that target CD79b(e.g., anti-CD79b immunoconjugates).

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

Provided herein are methods of treating a B-cell proliferative disorderin an individual comprising (a) an immunoconjugate comprising anantibody which binds CD79b linked to a cytotoxic agent and (b) anadditional therapeutic agent.

In particular, provided herein are methods for treating a B-cellproliferative disorder in an individual comprising administering to theindividual an effective amount of (a) an immunoconjugate comprising ananti-CD79b antibody linked to a cytotoxic agent and (b) an alkylatingagent. In some embodiments, provided herein are methods for treating aB-cell proliferative disorder in an individual comprising administeringto the individual an effective amount of (a) an immunoconjugatecomprising an anti-CD79b antibody linked to a cytotoxic agent, (b) ananti-CD20 antibody, and (c) an alkylating agent.

In some embodiments of any of the methods, the anti-CD20 antibody isrituximab. In some embodiments, rituximab is administered at about 375mg/m². In some embodiments of any of the methods, the anti-CD20 antibodyis a humanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1antibody is obinutuzumab. In some embodiments, obinutuzumab isadministered at about 1000 mg/m². In some embodiments of any of themethods, the anti-CD20 antibody is ofatumumab, ublituximab, and/oribritumomab tiuxetan.

In some embodiments of any of the methods, the alkylating agent is4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acidand salts thereof. In some embodiments of any of the methods, thealkylating agent is bendamustine. In some embodiments, bendamustine isadministered at about 25-120 mg/m². In some embodiments, bendamustine isadministered at about 90 mg/m².

In some embodiments of any of the methods, the cytotoxic agent is anantimitotic agent. In some embodiments, the antimitotic agent is aninhibitor of the polymerization of tubulin.

In some embodiments of any of the methods, the immunoconjugate has theformula Ab-(L-D)p, wherein: (a) Ab is the antibody which binds CD79b;(b) L is a linker; (c) D is the cytotoxic agent and the cytotoxic agentis selected from a maytansinoid or an auristatin; and (d) p ranges from1-8.

In some embodiments of any of the methods, D is an auristatin. In someembodiments of any of the methods, D has formula DE

and wherein R² and R⁶ are each methyl, R³ and R⁴ are each isopropyl, R⁵is H, R⁷ is sec-butyl, each R⁸ is independently selected from CH₃,O—CH₃, OH, and H; R⁹ is H; and R¹⁸ is —C(R⁸)₂—C(R⁸)₂-aryl. In someembodiments of any of the methods, D is MMAE.

In some embodiments of any of the methods, the linker is cleavable by aprotease. In some embodiments, the linker comprises a val-cit dipeptideor a Phe-homoLys dipeptide.

In some embodiments of any of the methods, the linker is acid-labile. Insome embodiments, the linker comprises hydrazone.

In some embodiments of any of the methods, the formula is:

wherein S is a sulfur atom.

In some embodiments of any of the methods, p ranges from 2-5.

In some embodiments of any of the methods, the antibody is a monoclonalantibody. In some embodiments, the antibody is a human, humanized, orchimeric antibody.

In some embodiments of any of the methods, the antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:23; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:26.

In some embodiments of any of the methods, the antibody comprises (a) aVH comprising the amino acid sequence of SEQ ID NO:19 and (b) a VLsequence comprises the amino acid sequence of SEQ ID NO:20. In someembodiments of any of the methods, the antibody comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO:36 and (b) a lightchain comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments of any of the methods, the antibody is a cysteineengineered antibody. In some embodiments, the antibody comprises anengineered cysteine at position 118 according to EU numbering conventionof the heavy chain (A118C). In some embodiments, the antibody comprisesan engineered cysteine at position 205 according to Kabat numberingconvention of the light chain (V205C). In some embodiments of any of themethods, the cytotoxic agent is linked to the anti-CD79b antibodythrough the engineered cysteine (e.g., at position 118 according to EUnumbering convention of the heavy chain and/or at position 205 accordingto Kabat numbering convention of the light chain). In some embodimentsof any of the methods, the antibody comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO:37 and (b) a light chaincomprising the amino acid sequence of SEQ ID NO:35. In some embodimentsof any of the methods, the antibody comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO:36 and (b) a light chaincomprising the amino acid sequence of SEQ ID NO:38.

In some embodiments of any of the methods, the B-cell proliferativedisorder is cancer. In some embodiments, the B-cell proliferativedisorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), or mantle cell lymphoma. In someembodiments, the B-cell proliferative disorder is NHL, such as indolentNHL and/or aggressive NHL. In some embodiments, the B-cell proliferativedisorder is indolent follicular lymphoma or diffuse large B-celllymphoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows change in tumor volume (mm³) upon treatment of WSU-CLCL2(Diffuse Large B-cell Lymphoma with (a) huMA79bv28-MC-vc-PAB-MMAE, (b)rituximab+bendamustine, and (c)huMA79bv28-MC-vc-PAB-MMAE+rituximab+bendamustine.huMA79bv28-MC-vc-PAB-MMAE: 2 mg/kg, iv, once on day 0; anti-CD20(rituximab): 30 mg/kg, ip, once on day 0, and bendamustine: 30 mg/kg,iv, once on day 0.

FIG. 2 shows change in tumor volume (mm³) upon treatment of tumorxenografts model of Granta-519 human mantle-cell lymphoma with (a)vehicle, (b) huMA79bv28-MC-vc-PAB-MMAE (DCDS4501A), (c) ABT-199, and (d)huMA79bv28-MC-vc-PAB-MMAE (DCDS4501A)+ABT-199. huMA79bv28-MC-vc-PAB-MMAE(DCDS4501A): 1 mg/kg, iv, once on day 0 and ABT-199: 100 mg/kg, po,qd21.

FIGS. 3A-3B show change in tumor volume (mm³) upon treatment of tumorxenografts model of WSU-DLCL2 (DLBCL) and TMD8 (ABC-DLBCL) with variouscombination therapy regimens including huMA79bv28-MC-vc-PAB-MMAE.

FIG. 4 shows change in tumor volume (mm³) upon treatment of tumorxenografts model of WSU-DLCL2 (DLBCL) with various combination therapyregimens huMA79bv28-MC-vc-PAB-MMAE.

DETAILED DESCRIPTION

Provided herein are methods of treating B-cell proliferative disorderssuch as indolent and aggressive NHL using combinations ofimmunoconjugates comprising an antibody which binds CD79b linked to acytotoxic agent (i.e., anti-CD79b immunoconjugate) and additionaltherapeutic agents, in particular, in some embodiments, theimmunoconjugates comprise an antimitotic agent such as an inhibitor ofthe polymerization of tubulin.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001).

II. Definitions

The term “CD79b”, as used herein, refers to any native CD79b from anyvertebrate source, including mammals such as primates (e.g., humans,cynomolgus monkey (cyno)) and rodents (e.g., mice and rats), unlessotherwise indicated. Human CD79b is also referred herein to as “Igβ,”“B29,” “DNA225786” or “PR036249.” An exemplary CD79b sequence includingthe signal sequence is shown in SEQ ID NO:1. An exemplary CD79b sequencewithout the signal sequence is shown in SEQ ID NO:2. The term “CD79b”encompasses “full-length,” unprocessed CD79b as well as any form ofCD79b that results from processing in the cell. The term alsoencompasses naturally occurring variants of CD79b, e.g., splicevariants, allelic variants and isoforms. The CD79b polypeptidesdescribed herein may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods. A “native sequence CD79b polypeptide” comprises apolypeptide having the same amino acid sequence as the correspondingCD79b polypeptide derived from nature. Such native sequence CD79bpolypeptides can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence CD79bpolypeptide” specifically encompasses naturally-occurring truncated orsecreted forms of the specific CD79b polypeptide (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of thepolypeptide.

“CD20” as used herein refers to the human B-lymphocyte antigen CD20(also known as CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5,and LF5; the sequence is characterized by the SwissProt database entryP11836) is a hydrophobic transmembrane protein with a molecular weightof approximately 35 kD located on pre-B and mature B lymphocytes.(Valentine, M. A., et al., J. Biol. Chem. 264(19) (1989 11282-11287;Tedder, T. F., et al, Proc. Nat. Acad. Sci. U.S.A. 85 (1988) 208-12;Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-80; Einfeld, D. A.et al., EMBO J. 7 (1988) 711-7; Tedder, T. F., et al., J. Immunol. 142(1989) 2560-8). The corresponding human gene is Membrane-spanning4-domains, subfamily A, member 1, also known as MS4A1. This gene encodesa member of the membrane-spanning 4A gene family. Members of thisnascent protein family are characterized by common structural featuresand similar intron/exon splice boundaries and display unique expressionpatterns among hematopoietic cells and nonlymphoid tissues. This geneencodes the B-lymphocyte surface molecule which plays a role in thedevelopment and differentiation of B-cells into plasma cells. Thisfamily member is localized to 11q12, among a cluster of family members.Alternative splicing of this gene results in two transcript variantswhich encode the same protein.

The terms “CD20” and “CD20 antigen” are used interchangeably herein, andinclude any variants, isoforms and species homologs of human CD20 whichare naturally expressed by cells or are expressed on cells transfectedwith the CD20 gene. Binding of an antibody of the invention to the CD20antigen mediate the killing of cells expressing CD20 (e.g., a tumorcell) by inactivating CD20. The killing of the cells expressing CD20 mayoccur by one or more of the following mechanisms: Cell death/apoptosisinduction, ADCC and CDC. Synonyms of CD20, as recognized in the art,include B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1,Leu-16, Bp35, BM5, and LF5.

The term “expression of the CD20” antigen is intended to indicate asignificant level of expression of the CD20 antigen in a cell, e.g., aT- or B-Cell. In one embodiment, patients to be treated according to themethods of this invention express significant levels of CD20 on a B-celltumor or cancer. Patients having a “CD20 expressing cancer” can bedetermined by standard assays known in the art. e.g., CD20 antigenexpression is measured using immunohistochemical (IHC) detection, FACSor via PCR-based detection of the corresponding mRNA.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “anti-CD79b antibody” or “an antibody that binds to CD79b”refers to an antibody that is capable of binding CD79b with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting CD79b. Preferably, the extent of bindingof an anti-CD79b antibody to an unrelated, non-CD79b protein is lessthan about 10% of the binding of the antibody to CD79b as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to CD79b has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, anti-CD79b antibodybinds to an epitope of CD79b that is conserved among CD79b fromdifferent species.

The term “anti-CD20 antibody” according to the invention refers to anantibody that is capable of binding CD20 with sufficient affinity suchthat the antibody is useful as a diagnostic and/or therapeutic agent intargeting CD20. Preferably, the extent of binding of an anti-CD20antibody to an unrelated, non-CD20 protein is less than about 10% of thebinding of the antibody to CD20 as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that binds to CD20 has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.In certain embodiments, anti-CD20 antibody binds to an epitope of CD20that is conserved among CD20 from different species.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T-cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1106 (nivolumab) described herein. In another specific aspect, aPD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. Inanother specific aspect, a PD-1 binding antagonist is CT-011(pidilizumab) described herein. In another specific aspect, a PD-1binding antagonist is AMP-224 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein. In still another specific aspect, an anti-PD-L1antibody is MEDI4736 described herein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific aspect, the PD-L2 bindingantagonist inhibits binding of PD-L2 to PD-1. In some embodiments, thePD-L2 antagonists include anti-PD-L2 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L2 witheither one or more of its binding partners, such as PD-1. In oneembodiment, a PD-L2 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L2 so as rendera dysfunctional T-cell less dysfunctional (e.g., enhancing effectorresponses to antigen recognition). In some embodiments, a PD-L2 bindingantagonist is an immunoadhesin.

The term “dysfunction” in the context of immune dysfunction, refers to astate of reduced immune responsiveness to antigenic stimulation. Theterm includes the common elements of both exhaustion and/or anergy inwhich antigen recognition may occur, but the ensuing immune response isineffective to control infection or tumor growth.

The term “dysfunctional”, as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into down-stream T-cell effectorfunctions, such as proliferation, cytokine production (e.g., IL-2)and/or target cell killing.

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g., increase in intracellular Ca⁺² in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of costimulation. The unresponsive state can oftenbe overridden by the presence of Interleukin-2. Anergic T-cells do notundergo clonal expansion and/or acquire effector functions.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, and/or 200%.The manner of measuring this enhancement is known to one of ordinaryskill in the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, a T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenicity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include treatmentwith a PD-1 axis binding antagonist and an anti-CD79b immunoconjugate(e.g., anti-CD79b-MC-vc-PAB-MMAE).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). The “variableregion” or “variable domain” of an antibody refers to the amino-terminaldomains of the heavy or light chain of the antibody. The variable domainof the heavy chain may be referred to as “VH.” The variable domain ofthe light chain may be referred to as “VL.” These domains are generallythe most variable parts of an antibody and contain the antigen-bindingsites.

“Isolated nucleic acid encoding an anti-CD79b antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: CIq binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B-cell receptor); and B-cellactivation.

“CD79b polypeptide variant” means a CD79b polypeptide, preferably anactive CD79b polypeptide, as defined herein having at least about 80%amino acid sequence identity with a full-length native sequence CD79bpolypeptide sequence as disclosed herein, a CD79b polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a CD79b polypeptide, with or without the signal peptide, as disclosedherein or any other fragment of a full-length CD79b polypeptide sequenceas disclosed herein (such as those encoded by a nucleic acid thatrepresents only a portion of the complete coding sequence for afull-length CD79b polypeptide). Such CD79b polypeptide variants include,for instance, CD79b polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the full-length nativeamino acid sequence. Ordinarily, a CD79b polypeptide variant will haveat least about 80% amino acid sequence identity, alternatively at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to afull-length native sequence CD79b polypeptide sequence as disclosedherein, a CD79b polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a CD79b polypeptide, withor without the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length CD79b polypeptidesequence as disclosed herein. Ordinarily, CD79b variant polypeptides areat least about 10 amino acids in length, alternatively at least about20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600 amino acids in length, or more. Optionally, CD79b variantpolypeptides will have no more than one conservative amino acidsubstitution as compared to the native CD79b polypeptide sequence,alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservativeamino acid substitution as compared to the native CD79b polypeptidesequence.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,B²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. More specificexamples include, but are not limited to, relapsed or refractory NHL,front line low grade NHL, Stage III/IV NHL, chemotherapy resistant NHL,precursor B lymphoblastic leukemia and/or lymphoma, small lymphocyticlymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocyticleukemia and/or small lymphocytic lymphoma, B-cell prolymphocyticlymphoma, immunocytoma and/or lymphoplasmacytic lymphoma,lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) large granularlymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin(cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentriclymphoma.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, reduction of free light chain, preventing occurrence or recurrenceof disease, alleviation of symptoms, diminishment of any direct orindirect pathological consequences of the disease, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the antibodiesdescribed herein are used to delay development of a disease or to slowthe progression of a disease.

The term “CD79b-positive cancer” refers to a cancer comprising cellsthat express CD79b on their surface. In some embodiments, expression ofCD79b on the cell surface is determined, for example, using antibodiesto CD79b in a method such as immunohistochemistry, FACS, etc.Alternatively, CD79b mRNA expression is considered to correlate to CD79bexpression on the cell surface and can be determined by a methodselected from in situ hybridization and RT-PCR (including quantitativeRT-PCR).

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeerlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®,Millennium Pharm.), disulfiram, epigallocatechin gallate,salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol,lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca),sunitinib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis),imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®,Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil),leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®,GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib(NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478,alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including topotecan and irinotecan);bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesinand bizelesin synthetic analogs); cryptophycins (particularlycryptophycin 1 and cryptophycin 8); adrenocorticosteroids (includingprednisone and prednisolone); cyproterone acetate; 5α-reductasesincluding finasteride and dutasteride); vorinostat, romidepsin,panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talcduocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, chlorophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosoureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl.Ed. Engl. 1994 33:183-186); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin),morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,everolimus, sotrataurin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folicacid analogs such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, testolactone;anti-adrenals such as aminoglutethimide, mitotane, trilostane; folicacid replenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfomithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR®(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE®(vinorelbine); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above; as well as combinations of two or moreof the above such as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone, andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovovin. Additional examplesinclude of chemotherapeutic agents include bendamustine (TREANDA®),ibrutinib, lenalidomide, and/or idelalisib (GS-1101).

Additional examples of chemotherapeutic agents include anti-hormonalagents that act to regulate, reduce, block, or inhibit the effects ofhormones that can promote the growth of cancer, and are often in theform of systemic, or whole-body treatment. They may be hormonesthemselves. Examples include anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON®); anti-progesterones; estrogen receptordown-regulators (ERDs); estrogen receptor antagonists such asfulvestrant (FASLODEX®); agents that function to suppress or shut downthe ovaries, for example, leutinizing hormone-releasing hormone (LHRH)agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelinacetate, buserelin acetate and tripterelin; anti-androgens such asflutamide, nilutamide and bicalutamide; and aromatase inhibitors thatinhibit the enzyme aromatase, which regulates estrogen production in theadrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMASIN®),formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), andanastrozole (ARIMIDEX®). In addition, such definition ofchemotherapeutic agents includes bisphosphonates such as clodronate (forexample, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095,zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);anti-sense oligonucleotides, particularly those that inhibit expressionof genes in signaling pathways implicated in aberrant cellproliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as THERATOPE®vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine,LEUVECTIN® vaccine, and VAXID® vaccine;

In some embodiments, the chemotherapeutic agent includes topoisomerase 1inhibitor (e.g., LURTOTECAN®); an anti-estrogen such as fulvestrant; aKit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinaseinhibitor); EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGFinhibitor such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX®);lapatinib and lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosinekinase small-molecule inhibitor also known as GW572016); 17AAG(geldanamycin derivative that is a heat shock protein (Hsp) 90 poison),and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

Chemotherapetuic agent also includes antibodies such as alemtuzumab(Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®,Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®,Genentech/Biogen Idec), ublituximab, ofatumumab, ibritumomab tiuxetan,pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additionalhumanized monoclonal antibodies with therapeutic potential as agents incombination with the compounds include: apolizumab, aselizumab,atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine,cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab,eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab,reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab,siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab,tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin,tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, andthe anti-interleukin-12 (ABT-874/J695, Wyeth Research and AbbottLaboratories) which is a recombinant exclusively human-sequence,full-length IgG1 λ antibody genetically modified to recognizeinterleukin-12 p40 protein.

As used herein, the term “cytokine” refers generically to proteinsreleased by one cell population that act on another cell asintercellular mediators or have an autocrine effect on the cellsproducing the proteins. Examples of such cytokines include lymphokines,monokines; interleukins (“ILs”) such as IL-1, IL-1α, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL 10, IL-11, IL-12, IL-13, IL-15,IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN®rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β, TGF-β1-3; andother polypeptide factors including leukemia inhibitory factor (“LIF”),ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”),cardiotrophin (“CT”), and kit ligand (“KL”).

As used herein, the term “chemokine” refers to soluble factors (e.g.,cytokines) that have the ability to selectively induce chemotaxis andactivation of leukocytes. They also trigger processes of angiogenesis,inflammation, wound healing, and tumorigenesis. Example chemokinesinclude IL-8, a human homolog of murine keratinocyte chemoattractant(KC).

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C1-C8 alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C1-C8 alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -and n-hexyl; whilebranched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₈ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H7), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., thealkyl moiety, including alkanyl, alkenyl or alkynyl groups, of thearylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14carbon atoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂₀R, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR,—C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X isindependently a halogen: F, C₁, Br, or I; and each R is independently—H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group orprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g., nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g.,polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.,polyethyleneamino, Jeffamine™); and diacid ester and amides includingsuccinate, succinamide, diglycolate, malonate, and caproamide. Invarious embodiments, linkers can comprise one or more amino acidresidues, such as valine, phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

It is understood that aspects and variations of the invention describedherein include “consisting of” and/or “consisting essentially of”aspects and variations.

A. Methods of Use

Provided herein are methods of treating a B-cell proliferative disorderin an individual comprising (a) an immunoconjugate comprising anantibody which binds CD79b linked to a cytotoxic agent and (b) anadditional therapeutic agent. In some embodiments, the additionaltherapeutic agent is a chemotherapeutic agent. In some embodiments, theadditional therapeutic agent is cytotoxic agent.

Provided herein are methods for treating a B-cell proliferative disorderin an individual comprising administering to the individual an effectiveamount of (a) an immunoconjugate comprising an anti-CD79b antibodylinked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate and (b) analkylating agent. In particular, provided herein are methods fortreating a B-cell proliferative disorder in an individual comprisingadministering to the individual an effective amount of (a) animmunoconjugate comprising an anti-CD79b antibody linked to a cytotoxicagent (i.e., anti-CD79b immunoconjugate), (b) an anti-CD20 antibody, and(c) an alkylating agent. In some embodiments, the anti-CD20 antibody isrituximab. In some embodiments, the anti-CD20 antibody is a humanizedB-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody isobinituzumab. In some embodiments, the anti-CD20 antibody is ofatumumab,ublituximab, and/or ibritumomab tiuxetan. In some embodiments, thealkylating agent is4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acidand salts thereof. In some embodiments, the alkylating agent isbendamustine. In some embodiments, the anti-CD79b immunoconjugate ishuMA79bv28-MC-vc-PAB-MMAE.

In addition, provided herein are methods for treating a B-cellproliferative disorder in an individual comprising administering to theindividual an effective amount of (a) an immunoconjugate comprising ananti-CD79b antibody linked to a cytotoxic agent (i.e., anti-CD79bimmunoconjugate) and (b) a BCL-2 inhibitor. In particular, providedherein are methods for treating a B-cell proliferative disorder in anindividual comprising administering to the individual an effectiveamount of (a) an immunoconjugate comprising an anti-CD79b antibodylinked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) ananti-CD20 antibody, and (c) a BCL-2 inhibitor. In some embodiments, theanti-CD20 antibody is rituximab. In some embodiments, the anti-CD20antibody is a humanized B-Ly1 antibody. In some embodiments, thehumanized B-Ly1 antibody is obinituzumab. In some embodiments, theanti-CD20 antibody is ofatumumab, ublituximab, and/or ibritumomabtiuxetan. In some embodiments, the BCL-2 inhibitor is4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamideand salts thereof. In some embodiments, the BCL-2 inhibitor isvenetoclax (CAS #: 1257044-40-8). In some embodiments, the anti-CD79bimmunoconjugate is huMA79bv28-MC-vc-PAB-MMAE.

Also provided herein are methods for treating a B-cell proliferativedisorder in an individual comprising administering to the individual aneffective amount of (a) an immunoconjugate comprising an anti-CD79bantibody linked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate)and (b) a phosphoinositide 3-kinase (PI3K) inhibitor. For example,provided herein are methods for treating a B-cell proliferative disorderin an individual comprising administering to the individual an effectiveamount of (a) an immunoconjugate comprising an anti-CD79b antibodylinked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) ananti-CD20 antibody, and (c) a phosphoinositide 3-kinase (PI3K)inhibitor. In some embodiments, the anti-CD20 antibody is rituximab. Insome embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody.In some embodiments, the humanized B-Ly1 antibody is obinituzumab. Insome embodiments, the anti-CD20 antibody is ofatumumab, ublituximab,and/or ibritumomab tiuxetan. In some embodiments, the PI3K inhibitorinhibits delta isoform PI3K (i.e., P1106). In some embodiments, the PI3Kinhibitor is5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinoneand salts thereof. In some embodiments, the PI3K inhibitor is idelalisib(CAS #: 870281-82-6). In some embodiments, the PI3K inhibitor inhibitsalpha and delta isoforms of PI3K. In some embodiments, the PI3Kinhibitor is2-{3-[2-(1-Isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamideand salts thereof. In some embodiments, the anti-CD79b immunoconjugateis huMA79bv28-MC-vc-PAB-MMAE.

Also provided herein are methods for treating a B-cell proliferativedisorder in an individual comprising administering to the individual aneffective amount of (a) an immunoconjugate comprising an anti-CD79bantibody linked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate)and (b) a Bruton's tyrosine kinase (BTK) inhibitor. In some embodiments,provided herein are methods for treating a B-cell proliferative disorderin an individual comprising administering to the individual an effectiveamount of (a) an immunoconjugate comprising an anti-CD79b antibodylinked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) ananti-CD20 antibody, and (c) a Bruton's tyrosine kinase (BTK) inhibitor.In some embodiments, the anti-CD20 antibody is rituximab. In someembodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody. Insome embodiments, the humanized B-Ly1 antibody is obinituzumab. In someembodiments, the anti-CD20 antibody is ofatumumab, ublituximab, and/oribritumomab tiuxetan. In some embodiments, the BTK inhibitor is1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-oneand salts thereof. In some embodiments, the BTK inhibitor is ibrutinib(CAS #: 936563-96-1). In some embodiments, the anti-CD79bimmunoconjugate is huMA79bv28-MC-vc-PAB-MMAE.

Provided herein are also methods for treating a B-cell proliferativedisorder in an individual comprising administering to the individual aneffective amount of (a) an immunoconjugate comprising an anti-CD79bantibody linked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate)and (b) thalidomide or a derivative thereof. For example, providedherein are methods for treating a B-cell proliferative disorder in anindividual comprising administering to the individual an effectiveamount of (a) an immunoconjugate comprising an anti-CD79b antibodylinked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) ananti-CD20 antibody, and (c) thalidomide or a derivative thereof. In someembodiments, the anti-CD20 antibody is rituximab. In some embodiments,the anti-CD20 antibody is a humanized B-Ly1 antibody. In someembodiments, the humanized B-Ly1 antibody is obinituzumab. In someembodiments, the anti-CD20 antibody is ofatumumab, ublituximab, and/oribritumomab tiuxetan. In some embodiments, the thalidomide or aderivative thereof is (RS)-3-(4-Amino-1-oxo1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione and salts thereof. Insome embodiments, the thalidomide or a derivative thereof islendalidomide (CAS #: 191732-72-6). In some embodiments, the anti-CD79bimmunoconjugate is huMA79bv28-MC-vc-PAB-MMAE.

Provided herein are also methods for treating a B-cell proliferativedisorder in an individual comprising administering to the individual aneffective amount of (a) an immunoconjugate comprising an anti-CD79bantibody linked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate)and (b) a PD-1 axis binding antagonist. For example, provided herein aremethods for treating a B-cell proliferative disorder in an individualcomprising administering to the individual an effective amount of (a) animmunoconjugate comprising an anti-CD79b antibody linked to a cytotoxicagent (i.e., anti-CD79b immunoconjugate), (b) an anti-CD20 antibody, and(c) a PD-1 axis binding agent. In some embodiments, the anti-CD20antibody is rituximab. In some embodiments, the anti-CD20 antibody is ahumanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1antibody is obinituzumab. In some embodiments, the anti-CD20 antibody isofatumumab, ublituximab, and/or ibritumomab tiuxetan. In someembodiments, the anti-CD79b immunoconjugate ishuMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the PD-1 axis bindingantagonist is selected from the group consisting of a PD-1 bindingantagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.PD-1 (programmed death 1) is also referred to in the art as “programmedcell death 1”, PDCD1, CD279 and SLEB2. PD-L1 (programmed death ligand 1)is also referred to in the art as “programmed cell death 1 ligand 1”,PDCD1LG1, CD274, B7-H, and PDL1. PD-L1 (programmed death ligand 1), alsoknown as PDL1, B7-H1, B7-4, CD274, and B7-H, is a transmembrane protein,and its interaction with PD-1 inhibits T-cell activation and cytokineproduction. PD-L2 (programmed death ligand 2) is also referred to in theart as “programmed cell death 1 ligand 2”, PDCD1LG2, CD273, B7-DC, Btdc,and PDL2. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1,PD-L1 and PD-L2. In some embodiments, the PD-1 axis binding antagonistis a PD-1 binding antagonist. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to its ligand binding partners.In some embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to PD-L2. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In someembodiments, the PD-1 binding antagonist is an antibody. In someembodiments, the PD-1 binding antagonist is selected from the groupconsisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab,lambrolizumab), CT-011 (pidilizumab), and AMP-224. Nivolumab, also knownas MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is ananti-PD-1 antibody described in WO2006/121168. In some embodiments, theanti-PD-1 antibody is Nivolumab (CAS Registry Number: 946414-94-4).Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab,KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described inWO2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is ananti-PD-1 antibody described in WO2009/101611. AMP-224, also known asB7-DCIg, is a PD-L2-Fc fusion soluble receptor described inWO2010/027827 and WO2011/066342. In some embodiments, the PD-1 axisbinding antagonist is a PD-L1 binding antagonist. In some embodiments,the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. Insome embodiments, the PD-L1 binding antagonist inhibits the binding ofPD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonistinhibits the binding of PD-L1 to both PD-1 and B7-1. In someembodiments, the PD-L1 binding antagonist is an antibody. In someembodiments, the PD-L1 binding antagonist is selected from the groupconsisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. AntibodyYW243.55.S70 is an anti-PD-L1 described in WO 2010/077634. MDX-1105,also known as BMS-936559, is an anti-PD-L1 antibody described inWO2007/005874. MEDI4736, is an anti-PD-L1 monoclonal antibody describedin WO2011/066389 and US2013/034559. Examples of anti-PDL1 antibodiesthat can be used in the methods described herein are described in PCTpatent application WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, whichare incorporated herein by reference. In some embodiments, the PD-1 axisbinding antagonist is an antibody. In some embodiments, the PD-1 axisbinding antagonist is a PD-L2 binding antagonist. In some embodiments,the PD-L2 binding antagonist is an antibody. In some embodiments, thePD-L2 binding antagonist is an immunoadhesin. In some embodiments, thecombination method enhances inhibition of tumor growth, increasedresponse rates and/or durable responses.

In some embodiments, an activating co-stimulatory molecule may includeCD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In someembodiments, the agonist directed against an activating co-stimulatorymolecule is an agonist antibody that binds to CD40, CD226, CD28, OX40,GITR, CD137, CD27, HVEM, or CD127. In some embodiments, method furthercomprises administration in conjunction with an antagonist directedagainst an inhibitory co-stimulatory molecule. In some embodiments, aninhibitory co-stimulatory molecule may include CTLA-4 (also known asCD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT,MICA/B, or arginase. In some embodiments, the antagonist directedagainst an inhibitory co-stimulatory molecule is an antagonist antibodythat binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4,IDO, TIGIT, MICA/B, or arginase. In some embodiments, method furthercomprises administration in conjunction with a treatment comprisingadoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL)expressing a chimeric antigen receptor (CAR).

In some embodiments of any of the methods, the cytotoxic agent is anantimitotic agent. Antimitotic agents are known in the art as well asinhibitors of the polymerization of tubulin. See e.g., Perez, Mol.Cancer Ther. 8:2086-2095 (2009), Doronina et al., Nat. Biotechnol.21:778-784 (2003), and Doronina et al., Bioconjug Chem. 17:114-124(2006). In some embodiments, the antimitotic agent includes, but is notlimited to, a maytansinoid, a dolastatin, an auristatin, and/or analogsand/or derivatives thereof. In some embodiments, the antimitotic agentis an auristatin and/or analog and/or derivative thereof. In someembodiments, the auristatin and/or analog and/or derivative thereof isMMAE. In some embodiments, the auristatin and/or analog and/orderivative thereof is MMAF.

In a further aspect, the invention provides for the use of an anti-CD79bimmunoconjugate in the manufacture or preparation of a medicament foruse in combination with an additional therapeutic agent. For example,provided herein is the use of an anti-CD79b immunoconjugate in themanufacture or preparation of a medicament for use in combination withan anti-CD20 antibody and an alkylating agent (e.g., bendamustine). Inone such embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent.

An “individual” according to any of the above embodiments may be ahuman.

In one embodiment, B-cell proliferative disease includes, but is notlimited to, lymphomas (e.g., B-Cell Non-Hodgkin's lymphomas (NHL)) andlymphocytic leukemias. Such lymphomas and lymphocytic leukemias includee.g. a) follicular lymphomas, b) Small Non-Cleaved CellLymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma,sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma), c) marginalzone lymphomas (including extranodal marginal zone B-cell lymphoma(Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginalzone B-cell lymphoma and splenic marginal zone lymphoma), d) Mantle celllymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse largecell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma, ImmunoblasticLymphoma, Primary Mediastinal B-Cell Lymphoma, AngiocentricLymphoma-Pulmonary B-Cell Lymphoma), f) hairy cell leukemia, g)lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/smalllymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasmacell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma,and/or j) Hodgkin's disease.

In some embodiments of any of the methods, the B-cell proliferativedisorder is cancer. In some embodiments, the B-cell proliferativedisorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), or mantle cell lymphoma. In someembodiments, the B-cell proliferative disorder is NHL, such as indolentNHL and/or aggressive NHL. In some embodiments, the B-cell proliferativedisorder is indolent follicular lymphoma or diffuse large B-celllymphoma (DLBCL). In some embodiments, the DLBCL is activated B cellDLBCL (ABC DLBCL). In some embodiments, the DLBCL is germinal centerB-cell like DLBCL (GCB DLBCL). In some embodiment, the DLBCL is BCL2positive (e.g., positive for BCL2 gene rearrangement,t(14;18)(q32;q21)). In some embodiments, the DLBCL is BCL2 negative(e.g., negative for BCL2 gene rearrangement, t(14;18)(q32;q21)).

In some embodiments of any of the methods, the B-cell proliferativedisorder is a histologically confirmed FL (Grade 1, 2, or 3a) or DLBCL.In some embodiments, the individual has received at least one priortherapy for FL or DLBCL. In some embodiments, the patient has receivedprior bendamustine and the duration must have been >1 year (for patientswho have relapse disease after a prior regimen). In some embodiments, atleast one bi-dimensionally measurable lesion on imaging scan definedas >1.5 cm in its longest dimension; confirmed availability of archivalor freshly collected tumor tissue meeting protocol-definedspecifications prior to study enrollment; Life expectancy of at least 24weeks; Eastern Cooperative Oncology Group (ECOG) Performance Status of0, 1, or 2; adequate hematological function; and/or, for women ofchildbearing potential, a negative serum pregnancy test result within 7days prior to commencement of dosing.

In some embodiments, the individual does not have a history of severeallergic or anaphylactic reactions to humanized or murine monoclonalantibodies (MAbs, or recombinant antibody-related fusion proteins) orknown sensitivity or allergy to murine products, contraindication tobendamustine, rituximab, or obinutuzumab. In some embodiments, theindividual does not have a history of sensitivity to mannitol, prior useof any MAb, radioimmunoconjugate, or antibody-drug conjugate (ADC)within 4 weeks before Cycle 1 Day 1, treatment with radiotherapy,chemotherapy, immunotherapy, immunosuppressive therapy, and/or anyinvestigational agent for the purposes of treating cancer within 2 weeksprior to Cycle 1 Day 1, ongoing corticosteroid use >30 mg/day prednisoneor equivalent, for purposes other than lymphoma symptom control,completion of autologous SCT within 100 days prior to Cycle 1 Day 1,prior allogeneic SCT, eligibility for autologous SCT (patients withrelapsed/refractory DLBCL), Grade 3b FL, history of transformation ofindolent disease to DLBCL, primary CNS lymphoma, current Grade >1peripheral neuropathy, evidence of significant, uncontrolled concomitantdiseases that could affect compliance with the protocol orinterpretation of results, including significant cardiovascular disease(such as New York Heart Association Class III or IV cardiac disease,myocardial infarction within the last 6 months, unstable arrhythmias, orunstable angina) or significant pulmonary disease (including obstructivepulmonary disease and history of bronchospasm), known active bacterial,viral, fungal, mycobacterial, parasitic, or other infection (excludingfungal infections of nail beds) at study enrollment or any major episodeof infection requiring treatment with intravenous (IV) antibiotics orhospitalization within 4 weeks prior to Cycle 1 Day 1, patients withsuspected or latent tuberculosis, positive test results for chronichepatitis B virus (HBV) infection or for hepatitis C virus (HCV)antibody, known infection with HIV or human T-cell leukemia virus 1(HTLV-1) virus, women who are pregnant or lactating or who intend tobecome pregnant within a year of the last dose of rituximab orobinutuzumab, and/or evidence of laboratory abnormalities in standardrenal, hepatic or coagulation function tests.

In some embodiments of any of the methods, the B-cell proliferativedisorder is a relapsed or refractory B-cell proliferative disorder. Insome embodiments, relapsed or refractory B-cell proliferative disorderas used herein includes patients who have received at least 1 priorchemotherapy containing treatment regimen. In some embodiments, relapsedpatients generally have developed progressive disease following aresponse to the prior chemotherapy-containing treatment regimen. In someembodiments, refractory patients have generally failed to respond orrelapsed within 6 months to the last prior chemotherapy-containingregimen. In some embodiments, relapsed/refractory follicular lymphoma(FL) patients who have relapsed to prior regimen(s) after having adocumented history of response (complete response [CR], CR unconfirmed[CRu], or partial response [PR]) of >/=6 months in duration fromcompletion of regimen(s); refractory to any prior regimen, defined as noresponse to the prior therapy, or progression within 6 months ofcompletion of the last dose of therapy. In some embodiments,relapsed/refractory DLBCL patients are patients who are ineligible forsecond-line stem cell transplant (SCT), with progressive disease or noresponse (stable disease [SD])<6 months from start of initial therapy;patients who are ineligible for second-line SCT, with disease relapseafter initial response of >/=6 months from start of initial therapy;patients who are ineligible for third-line (or beyond) SCT, withprogressive disease or no response (SD)<6 months from start of priortherapy; patients who are ineligible for third-line (or beyond) SCT withdisease relapse after initial response of >/=6 months from start ofprior therapy.

In some embodiments, the individual having a B-cell proliferativedisorder is previously untreated. In some embodiments, previouslyuntreated as used herein includes patients diagnosed with a B-cellproliferative disease, but who have, in general, received no priorchemotherapy or immunotherapy. Patients with a history of emergency,loco-regional radiotherapy (e.g., for relief of compressive signs orsymptoms) or corticosteroids can still be considered previouslyuntreated.

An immunoconjugate provided herein (and any additional therapeuticagent) for use in any of the therapeutic methods described herein can beadministered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.,by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

In some embodiments of any of the methods, if the administration isintravenous the initial infusion time for the anti-CD79b immunoconjugateor the additional therapeutic agent may be longer than subsequentinfusion times, for instance approximately 90 minutes for the initialinfusion, and approximately 30 minutes for subsequent infusions (if theinitial infusion is well tolerated).

The terms “co-administration” or “co-administering” refer to theadministration of the anti-CD79b immunoconjugate and the additionaltherapeutic agent as two separate formulations (or as one singleformulation). The co-administration can be simultaneous or sequential ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities. Theanti-CD79b immunoconjugate and the additional therapeutic agent areco-administered either simultaneously or sequentially. In someembodiments, when both therapeutic agents are co-administeredsequentially the dose is administered either on the same day in twoseparate administrations, or one of the agents is administered on day 1and the second is co-administered on day 2 to day 7, preferably on day 2to 4. Thus in one embodiment the term “sequentially” means within 7 daysafter the dose of the first component, preferably within 4 days afterthe dose of the first component; and the term “simultaneously” means atthe same time. The term “co-administration” with respect to themaintenance doses of said the anti-CD79b immunoconjugate and theadditional therapeutic agent means that the maintenance doses can beeither co-administered simultaneously, if the treatment cycle isappropriate for both drugs, e.g., every week. Or anti-CD79bimmunoconjugate is e.g., administered e.g., every first to third day andthe additional therapeutic is administered every week. Or themaintenance doses are co-administered sequentially, either within one orwithin several days.

Anti-CD79b immunoconjugates and additional therapeutic agents providedherein for use in any of the therapeutic methods described herein wouldbe formulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The immunoconjugate need not be, but isoptionally formulated with one or more agents currently used to preventor treat the disorder in question.

The amount of co-administration of the anti-CD79b immunoconjugate andthe additional therapeutic agent and the timing of co-administrationwill depend on the type (species, gender, age, weight, etc.) andcondition of the patient being treated and the severity of the diseaseor condition being treated. The anti-CD79b immunoconjugate and theadditional therapeutic agent are suitably co-administered to the patientat one time or over a series of treatments e.g., on the same day or onthe day after.

In some embodiments of any of the methods, the dosage of anti-CD79bimmunoconjugate (such as huMA79bv28-MC-vc-PAB-MMAE) is between about anyof 1.4-5 mg/kg, 1.8-4 mg/kg, 1.8-3.2 mg/kg, and/or 1.8-2.4 mg/kg. Insome embodiments of any of the methods, the dosage of anti-CD79immunoconjugate is about any of 1.4, 1.8, 2.0, 2.2, 2.4, 2.8, 3.2, 3.6,4.0, 4.4, and/or 4.8 mg/kg. In some embodiments, the dosage ofanti-CD79b immunoconjugate is about 1.8 mg/kg. In some embodiments, thedosage of anti-CD79b immunoconjugate is about 2.4 mg/kg. In someembodiments, the dosage of anti-CD79b immunoconjugate is about 3.2mg/kg. In some embodiments, the dosage of anti-CD79b immunoconjugate isabout 3.6 mg/kg. In some embodiments of any of the methods, theanti-CD79b immunoconjugate is administered q3wk. In some embodiments,the anti-CD79b immunoconjugate is administered via intravenous infusion.The dosage administered via infusion is in the range of about 1 μg/m2 toabout 10,000 μg/m2 per dose, generally one dose per week for a total ofone, two, three or four doses. Alternatively, the dosage range is ofabout 1 μg/m2 to about 1000 μg/m2, about 1 μg/m2 to about 800 μg/m2,about 1 μg/m2 to about 600 μg/m2, about 1 μg/m2 to about 400 μg/m2,about 10 μg/m2 to about 500 μg/m2, about 10 μg/m2 to about 300 μg/m2,about 10 μg/m2 to about 200 μg/m2, and about 1 μg/m2 to about 200 μg/m2.The dose may be administered once per day, once per week, multiple timesper week, but less than once per day, multiple times per month but lessthan once per day, multiple times per month but less than once per week,once per month or intermittently to relieve or alleviate symptoms of thedisease. Administration may continue at any of the disclosed intervalsuntil remission of the tumor or symptoms of the lymphoma, leukemia beingtreated. Administration may continue after remission or relief ofsymptoms is achieved where such remission or relief is prolonged by suchcontinued administration.

In some embodiments of any of the methods, the dosage of the anti-CD20antibody is between about 300-1600 mg/m² and/or 300-2000 mg In someembodiments of any of the methods, the dosage of the anti-CD20 antibodyis about any of 300, 375, 600, 1000, or 1250 mg/m² and/or 300, 1000, or2000 mg. In some embodiments, the anti-CD20 antibody is rituximab andthe dosage administered is 375 mg/m². In some embodiments, the anti-CD20antibody is obinutuzumab and the dosage administered is 1000 mg/m². Insome embodiments, the anti-CD20 antibody is administered q3wk. In someembodiments, the dosage of said afucosylated anti-CD20 antibody(preferably the afucosylated humanized B-Ly1 antibody) may be 800 to1600 mg (in one embodiment 800 to 1200 mg) on day 1, 8, 15 of a 3- to6-weeks-dosage-cycle and then in a dosage of 400 to 1200 (in oneembodiment 800 to 1200 mg) on day 1 of up to nine 3- to4-weeks-dosage-cycles. In some embodiments, the dose is a flat dose 1000mg in a three-weeks-dosage schedule, with the possibility of anadditional cycle of a flat dose of 1000 mg in the second week.

Exemplary dosing regimens for the combination therapy of anti-CD79bimmunoconjugates (such as huMA79bv28-MC-vc-PAB-MMAE) and other agentsinclude, but are not limited to, anti-CD79 immunoconjugate (such ashuMA79bv28-MC-vc-PAB-MMAE) administered at about 1.4-5 mg/kg q3wk, plus375 mg/m² q3wk rituximab, and 25-120 mg/m² bendamustine (e.g.,bendamustine hydrochloride) dl and 2 of q3wk daily. In some embodiments,the anti-CD79 immunoconjugate is administered at about any of 1.8 mg/kg,2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 3.2 mg/kg, or 4.0 mg/kg. In someembodiments, the anti-CD79b immunoconjugate is administered at about 1.8mg/kg. In some embodiments, the anti-CD79b immunoconjugate isadministered at about 2.4 mg/kg. In some embodiments, bendamustine isadministered at about 90 mg/m².

Another exemplary dosage regime for the combination therapy ofanti-CD79b immunoconjugates (such as huMA79bv28-MC-vc-PAB-MMAE) andother agents include, but are not limited to, anti-CD79 immunoconjugate(such as huMA79bv28-MC-vc-PAB-MMAE) administered 1.4-5 mg/kg q3wk, plus1000 mg/m² q3wk obinutuzumab, and 25-120 mg/m² bendamustine dl and 2 ofq3wk daily. In some embodiments, the anti-CD79 immunoconjugate isadministered at about any of 1.8 mg/kg, 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg,3.2 mg/kg, or 4.0 mg/kg. In some embodiments, the anti-CD79bimmunoconjugate is administered at about 1.8 mg/kg. In some embodiments,the anti-CD79b immunoconjugate is administered at about 2.4 mg/kg. Insome embodiments, bendamustine is administered at about 90 mg/m².

B. Agents for Use in the Methods Described Herein

Provided herein are anti-CD79b immunoconjugates and additionaltherapeutic agents for use in the methods described herein. In someembodiments, the additional therapeutic agent is an antibody. In someembodiments, the additional therapeutic agent is a small molecule. Insome embodiments, the additional therapeutic agent is an anti-CD20antibody and bendamustine.

1. Anti-CD79b Immunoconjugates Comprising Anti-CD79b Antibodies andOther Embodiments

Provided herein are anti-CD79b antibodies for the anti-CD79bimmunoconjugates for use in the methods described herein comprisinganti-CD79b immunoconjugates and an additional therapeutic agent.

In some embodiments, the methods herein provide an immunoconjugatecomprising an anti-CD79b antibody comprising at least one, two, three,four, five, or six HVRs selected from (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 23; (d) HVR-L1 comprising an amino acid sequence of SEQ IDNO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. Insome such embodiments, the immunoconjugate comprises at least one of:(i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, and/or(ii) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 24.

In some embodiments, provided herein for use in the methods areimmunoconjugates comprising an anti-CD79b antibody comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1comprising an amino acid sequence of SEQ ID NO: 24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 26. In some suchembodiments, the immunoconjugate comprises at least one of: (i) HVR-H3comprising the amino acid sequence of SEQ ID NO: 23, and/or (ii) HVR-L1comprising the amino acid sequence of SEQ ID NO:24. In one aspect,provided herein are immunoconjugates comprising an anti-CD79bimmunoconjugate comprising at least one, at least two, or all three VHHVR sequences selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 22; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 23. In some embodiments, the immunoconjugate comprises HVR-H3comprising the amino acid sequence of SEQ ID NO: 23. In anotherembodiment, the immunoconjugate comprises HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 23 and HVR-L3 comprising the amino acidsequence of SEQ ID NO: 26. In a further embodiment, the immunoconjugatecomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23,HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and HVR-H2comprising the amino acid sequence of SEQ ID NO: 22. In a furtherembodiment, the immunoconjugate comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 22; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 23.

In another aspect, the immunoconjugate comprises an anti-CD79b antibodycomprising at least one, at least two, or all three VL HVR sequencesselected from (a) HVR-L1 comprising an amino acid sequence of SEQ ID NO:24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. Inanother aspect, provided herein are immunoconjugates comprising at leastone, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 26. In one embodiment,the immunoconjugate comprises (a) HVR-L1 comprising an amino acidsequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 26. In some embodiments, the immunoconjugate comprises anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. In someembodiments, the immunoconjugate comprises an HVR-L1 comprising theamino acid sequence of SEQ ID NO: 24. In some embodiments, theimmunoconjugate comprises (a) HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO: 26.

In another aspect, the immunoconjugate comprises an anti-CD79b antibodycomprising (a) a VH domain comprising at least one, at least two, or allthree VH HVR sequences selected from (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 21, (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 22, and (iii) HVR-H3 comprising an amino acidsequence selected from SEQ ID NO:23; and (b) a VL domain comprising atleast one, at least two, or all three VL HVR sequences selected from (i)HVR-L1 comprising an amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 25, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO: 26. In some suchembodiments, the immunoconjugate comprises at least one of: (i) HVR-H3comprising the amino acid sequence of SEQ ID NO: 23, and/or (ii) HVR-L1comprising the amino acid sequence of SEQ ID NO: 24.

In another aspect, the provided herein are immunoconjugates comprising(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1comprising an amino acid sequence of SEQ ID NO: 24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 26. In some suchembodiments, the immunoconjugate comprises at least one of: HVR-H3comprising the amino acid sequence of SEQ ID NO: 23 and/or HVR-L1comprising an amino acid sequence of SEQ ID NO: 24. In another aspect,provided are immunoconjugates comprising (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In any of the above embodiments, the anti-CD79b immunoconjugatescomprises a humanized anti-CD79b antibody. In one embodiment, ananti-CD79b antibody comprises HVRs as in any of the above embodiments,and further comprises a human acceptor framework, e.g., a humanimmunoglobulin framework or a human consensus framework. In certainembodiments, the human acceptor framework is the human VL kappa 1(VL_(KI)) framework and/or the VH framework VH_(III). In someembodiments, a humanized anti-CD79b antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1comprising an amino acid sequence of SEQ ID NO: 24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a humanized anti-CD79b antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 26.

In another aspect, an anti-CD79b immunoconjugate comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 19. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 19 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD79b immunoconjugate comprising thatsequence retains the ability to bind to CD79b. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 19. In certain embodiments, a total of 1 to 5amino acids have been substituted, inserted and/or deleted in SEQ ID NO:19. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs).

Optionally, the anti-CD79b immunoconjugate comprises the VH sequence ofany one of SEQ ID NO: 19, including post-translational modifications ofthat sequence. In some embodiments, the anti-CD79b immunoconjugatecomprises the VH sequence of SEQ ID NO: 19, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 21, (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 22, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23.

In some embodiments, an anti-CD79b immunoconjugate comprises a lightchain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO: 20. In certain embodiments, a VL sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to theamino acid sequence of SEQ ID NO: 20 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD79b immunoconjugate comprising thatsequence retains the ability to bind to CD79b. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 20. In certain embodiments, a total of 1 to 5amino acids have been substituted, inserted and/or deleted in SEQ ID NO:20. In certain embodiments, the substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-CD79b immunoconjugate comprises the VL sequence of any one of SEQID NO: 20, including post-translational modifications of that sequence.In some embodiments, the anti-CD79b immunoconjugate comprises the VLsequence of SEQ ID NO: 20, including post-translational modifications ofthat sequence. In a particular embodiment, the VL comprises one, two orthree HVRs selected from (a) HVR-L1 comprising an amino acid sequence ofSEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the VL comprises one, two or three HVRsselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In another aspect, an anti-CD79b immunoconjugate comprises a VH as inany of the embodiments provided above, and a VL as in any of theembodiments provided above. In some embodiments, the antibody comprisesthe VH and VL sequences in SEQ ID NO: 19 and SEQ ID NO: 20,respectively, including post-translational modifications of thosesequences.

In a further aspect, provided herein are anti-CD79b immunoconjugatesthat binds to the same epitope as an anti-CD79b antibody providedherein. For example, in certain embodiments, immunoconjugate is providedthat binds to the same epitope as an anti-CD79b antibody comprising a VHsequence of SEQ ID NO: 19 and a VL sequence of SEQ ID NO: 20.

In a further aspect of the invention, an anti-CD79b immunoconjugateaccording to any of the above embodiments comprises a monoclonalantibody, including a chimeric, humanized or human antibody. In oneembodiment, an anti-CD79b immunoconjugate comprises an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the immunoconjugate comprises a substantially fulllength antibody, e.g., an IgG1 antibody or other antibody class orisotype as defined herein.

2. Anti-CD20 Antibodies and Other Embodiments

Provided herein are anti-CD20 antibodies for use in the methodsdescribed herein comprising anti-CD79b immunoconjugates and anadditional therapeutic agent.

Depending on binding properties and biological activities of anti-CD20antibodies to the CD20 antigen, two types of anti-CD20 antibodies (typeI and type II anti-CD20 antibodies) can be distinguished according toCragg, M. S., et al., Blood 103 (2004) 2738-2743; and Cragg, M. S., etal., Blood 101 (2003) 1045-1052, see Table 1.

TABLE 1 Properties of type I and type II anti-CD20 antibodies Type Ianti-CD20 antibodies type II anti-CD20 antibodies type I CD20 epitopetype II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20to lipid rafts Increased CDC (if IgG1 isotype) Decreased CDC (if IgG1isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype)Full binding capacity Reduced binding capacity Homotypic aggregationStronger homotypic aggregation Apoptosis induction upon Strong celldeath induction cross-linking without cross-linking

Examples of type I anti-CD20 antibodies include e.g., rituximab, HI47IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005/103081), 2F2IgG1 (as disclosed and WO 2004/035607 and WO 2005/103081) and 2H7 IgG1(as disclosed in WO 2004/056312). In some embodiments, the anti-CD20antibody is rituximab antibody. In some embodiments, the rituximabantibody (reference antibody; example of a type I anti-CD20 antibody) isa genetically engineered chimeric human gamma 1 murine constant domaincontaining monoclonal antibody directed against the human CD20 antigen.However, this antibody is not glycoengineered and not afucosylated andthus has an amount of fucose of at least 85%. This chimeric antibodycontains human gamma 1 constant domains and is identified by the name“C2B8” in U.S. Pat. No. 5,736,137 (Andersen, et. al.) issued on Apr. 17,1998, assigned to IDEC Pharmaceuticals Corporation. Rituximab isapproved for the treatment of patients with relapsed or refractinglow-grade or follicular, CD20 positive, B-cell non-Hodgkin's lymphoma.In vitro mechanism of action studies have shown that rituximab exhibitshuman complement-dependent cytotoxicity (CDC) (Reff, M. E., et. al,Blood 83(2) (1994) 435-445). Additionally, it exhibits activity inassays that measure antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the anti-CD20 antibodies are an afucosylatedanti-CD20 antibody.

Examples of type II anti-CD20 antibodies include e.g., humanized B-Ly1antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1.Typically type II anti-CD20 antibodies of the IgG1 isotype showcharacteristic CDC properties. Type II anti-CD20 antibodies have adecreased CDC (if IgG1 isotype) compared to type I antibodies of theIgG1 isotype. In one embodiment said type II anti-CD20 antibody, e.g., aGA101 antibody, has increased antibody dependent cellular cytotoxicity(ADCC). In some embodiments, the type II anti-CD20 antibodies, morepreferably an afucosylated humanized B-Ly1 antibody as described in WO2005/044859 and WO 2007/031875.

In some embodiments, the anti-CD20 antibody is GA101 antibody. In someembodiments, the GA101 antibody as used herein refers to any one of thefollowing antibodies that bind human CD20: (1) an antibody comprising anHVR-H1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-H2comprising the amino acid sequence of SEQ ID NO:6, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:7, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:8, an HVR-L2 comprising the amino acidsequence of SEQ ID NO:9, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:10; (2) an antibody comprising a VH domaincomprising the amino acid sequence of SEQ ID NO:11 and a VL domaincomprising the amino acid sequence of SEQ ID NO:12, (3) an antibodycomprising an amino acid sequence of SEQ ID NO:13 and an amino acidsequence of SEQ ID NO: 14; (4) an antibody known as obinutuzumab, or (5)an antibody that comprises an amino acid sequence that has at least 95%,96%, 97%, 98% or 99% sequence identity with amino acid sequence of SEQID NO:13 and that comprises an amino acid sequence that has at least95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequenceof SEQ ID NO: 14. In one embodiment, the GA101 antibody is an IgG1isotype antibody.

In some embodiments, the anti-CD20 antibody is a humanized B-Ly1antibody. In some embodiments, the humanized B-Ly1 antibody refers tohumanized B-Ly1 antibody as disclosed in WO 2005/044859 and WO2007/031875, which were obtained from the murine monoclonal anti-CD20antibody B-Ly1 (variable region of the murine heavy chain (VH): SEQ IDNO: 3; variable region of the murine light chain (VL): SEQ ID NO: 4—seePoppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) bychimerization with a human constant domain from IgG1 and followinghumanization (see WO 2005/044859 and WO 2007/031875). The humanizedB-Ly1 antibodies are disclosed in detail in WO 2005/044859 and WO2007/031875.

In one embodiment, the humanized B-Ly1 antibody has variable region ofthe heavy chain (VH) selected from group of SEQ ID NO:15-16 and 40-55(corresponding to B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO 2005/044859and WO 2007/031875). In one specific embodiment, such variable domain isselected from the group consisting of SEQ ID NO: 15, 16, 42, 44, 46, 48and 50 (corresponding to B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 andB-HL13 of WO 2005/044859 and WO 2007/031875). In one specificembodiment, the humanized B-Ly1 antibody has variable region of thelight chain (VL) of SEQ ID NO:55 (corresponding to B-KV1 of WO2005/044859 and WO 2007/031875). In one specific embodiment, thehumanized B-Ly1 antibody has a variable region of the heavy chain (VH)of SEQ ID NO:42 (corresponding to B-HH6 of WO 2005/044859 and WO2007/031875) and a variable region of the light chain (VL) of SEQ IDNO:55 (corresponding to B-KV1 of WO 2005/044859 and WO 2007/031875).Furthermore in one embodiment, the humanized B-Ly1 antibody is an IgG1antibody. According to the invention, such afocusylated humanized B-Ly1antibodies are glycoengineered (GE) in the Fc region according to theprocedures described in WO 2005/044859, WO 2004/065540, WO 2007/031875,Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and WO 99/154342.In one embodiment, the afucosylated glyco-engineered humanized B-Ly1 isB-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody isobinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4,2012, p. 453). As used herein, obinutuzumab is synonymous for GA101 orRO5072759. This replaces all previous versions (e.g., Vol. 25, No. 1,2011, p. 75-76), and is formerly known as afutuzumab (recommended INN,WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2,2008, p. 124). In some embodiments, the humanized B-Ly1 antibody is anantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO:17 and a light chain comprising the amino acid sequence of SEQID NO:18 or an antigen-binding fragment thereof. In some embodiments,the humanized B-Ly1 antibody comprises a heavy chain variable regioncomprising the three heavy chain CDRs of SEQ ID NO:17 and a light chainvariable region comprising the three light chain CDRs of SEQ ID NO:18.

In some embodiments, the humanized B-Ly1 antibody is an afucosylatedglyco-engineered humanized B-Ly1. Such glycoengineered humanized B-Ly1antibodies have an altered pattern of glycosylation in the Fc region,preferably having a reduced level of fucose residues. Preferably theamount of fucose is about 60% or less of the total amount ofoligosaccharides at Asn297 (in one embodiment the amount of fucose isbetween about 40% and about 60%, in another embodiment the amount offucose is about 50% or less, and in still another embodiment the amountof fucose is about 30% or less). Furthermore the oligosaccharides of theFc region are preferably bisected. These glycoengineered humanized B-Ly1antibodies have an increased ADCC.

The “ratio of the binding capacities to CD20 on Raji cells (ATCC-No.CCL-86) of an anti-CD20 antibodies compared to rituximab” is determinedby direct immunofluorescence measurement (the mean fluorescenceintensities (MFI) is measured) using said anti-CD20 antibody conjugatedwith Cy5 and rituximab conjugated with Cy5 in a FACSArray (BectonDickinson) with Raji cells (ATCC-No. CCL-86), as described in ExampleNo. 2, and calculated as follows:

${{Ratio}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{binding}\mspace{14mu}{capacities}\mspace{14mu}{to}\mspace{14mu}{CD20}\mspace{14mu}{on}\mspace{14mu}{Raji}\mspace{14mu}{{cells}\left( {{ATCC}\text{-}{{No}.\mspace{14mu}{CCL}}\text{-}86} \right)}} = {\frac{{MFI}\left( {{Cy5}\text{-}{anti}\text{-}{CD20}\mspace{14mu}{antibody}} \right)}{{MFI}\left( {{Cy5}\text{-}{rituximab}} \right)} \times \frac{{Cy5}\text{-}{labeling}\mspace{14mu}{{ratio}\left( {{Cy5}\text{-}{rituximab}} \right)}}{{Cy5}\text{-}{labeling}\mspace{14mu}{{ratio}\left( {{Cy5}\text{-}{anti}\text{-}{CD20}\mspace{14mu}{antibody}} \right)}}}$

MFI is the mean fluorescent intensity. The “Cy5-labeling ratio” as usedherein means the number of Cy5-label molecules per molecule antibody.

Typically said type II anti-CD20 antibody has a ratio of the bindingcapacities to CD20 on Raji cells (ATCC-No. CCL-86) of said secondanti-CD20 antibody compared to rituximab of 0.3 to 0.6, and in oneembodiment, 0.35 to 0.55, and in yet another embodiment, 0.4 to 0.5.

By “antibody having increased antibody dependent cellular cytotoxicity(ADCC)”, it is meant an antibody, as that term is defined herein, havingincreased ADCC as determined by any suitable method known to those ofordinary skill in the art.

One accepted in vitro ADCC assay is as follows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen-binding region of the        antibody;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:        -   i) the PBMCs are isolated using standard density            centrifugation procedures and are suspended at 5×10⁶            cells/ml in RPMI cell culture medium;        -   ii) the target cells are grown by standard tissue culture            methods, harvested from the exponential growth phase with a            viability higher than 90%, washed in RPMI cell culture            medium, labeled with 100 micro-Curies of ⁵¹Cr, washed twice            with cell culture medium, and resuspended in cell culture            medium at a density of 10⁵ cells/ml;        -   iii) 100 microliters of the final target cell suspension            above are transferred to each well of a 96-well microtiter            plate;        -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04            ng/ml in cell culture medium and 50 microliters of the            resulting antibody solutions are added to the target cells            in the 96-well microtiter plate, testing in triplicate            various antibody concentrations covering the whole            concentration range above;        -   v) for the maximum release (MR) controls, 3 additional wells            in the plate containing the labeled target cells, receive 50            microliters of a 2% (VN) aqueous solution of non-ionic            detergent (Nonidet, Sigma, St. Louis), instead of the            antibody solution (point iv above);        -   vi) for the spontaneous release (SR) controls, 3 additional            wells in the plate containing the labeled target cells,            receive 50 microliters of RPMI cell culture medium instead            of the antibody solution (point iv above);        -   vii) the 96-well microtiter plate is then centrifuged at            50×g for 1 minute and incubated for 1 hour at 4° C.; viii)            50 microliters of the PBMC suspension (point i above) are            added to each well to yield an effector:target cell ratio of            25:1 and the plates are placed in an incubator under 5% CO₂            atmosphere at 37° C. for 4 hours;        -   ix) the cell-free supernatant from each well is harvested            and the experimentally released radioactivity (ER) is            quantified using a gamma counter;        -   x) the percentage of specific lysis is calculated for each            antibody concentration according to the formula            (ER-MR)/(MR-SR)×100, where ER is the average radioactivity            quantified (see point ix above) for that antibody            concentration, MR is the average radioactivity quantified            (see point ix above) for the MR controls (see point V            above), and SR is the average radioactivity quantified (see            point ix above) for the SR controls (see point vi above);    -   4) “increased ADCC” is defined as either an increase in the        maximum percentage of specific lysis observed within the        antibody concentration range tested above, and/or a reduction in        the concentration of antibody required to achieve one half of        the maximum percentage of specific lysis observed within the        antibody concentration range tested above. In one embodiment,        the increase in ADCC is relative to the ADCC, measured with the        above assay, mediated by the same antibody, produced by the same        type of host cells, using the same standard production,        purification, formulation and storage methods, which are known        to those skilled in the art, except that the comparator antibody        (lacking increased ADCC) has not been produced by host cells        engineered to overexpress GnTIII and/or engineered to have        reduced expression from the fucosyltransferase 8 (FUT8) gene        (e.g., including, engineered for FUT8 knock out).

In some embodiments, the “increased ADCC” can be obtained by, forexample, mutating and/or glycoengineering of said antibodies. In oneembodiment, the antibody is glycoengineered to have a biantennaryoligosaccharide attached to the Fc region of the antibody that isbisected by GlcNAc. In another embodiment, the antibody isglycoengineered to lack fucose on the carbohydrate attached to the Fcregion by expressing the antibody in a host cell that is deficient inprotein fucosylation (e.g., Lec13 CHO cells or cells having analpha-1,6-fucosyltransferase gene (FUT8) deleted or the FUT geneexpression knocked down). In yet another embodiment, the antibodysequence has been engineered in its Fc region to enhance ADCC (e.g., inone embodiment, such engineered antibody variant comprises an Fc regionwith one or more amino acid substitutions at positions 298, 333, and/or334 of the Fc region (EU numbering of residues)).

In some embodiments, the term “complement-dependent cytotoxicity (CDC)”refers to lysis of human tumor target cells by the antibody according tothe invention in the presence of complement. CDC can be measured by thetreatment of a preparation of CD20 expressing cells with an anti-CD20antibody according to the invention in the presence of complement. CDCis found if the antibody induces at a concentration of 100 nM the lysis(cell death) of 20% or more of the tumor cells after 4 hours. In oneembodiment, the assay is performed with ⁵¹Cr or Eu labeled tumor cellsand measurement of released ⁵¹Cr or Eu. Controls include the incubationof the tumor target cells with complement but without the antibody.

In a further aspect of the invention, an anti-CD20 antibody according toany of the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD20 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a substantially full lengthantibody, e.g., an IgG1 antibody, IgG2a antibody or other antibody classor isotype as defined herein.

In a further aspect, an antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM, and optionally is ≥10⁻¹³ M. (e.g., 10⁻⁸ M orless, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²¹I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)k_(on) See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds10⁶M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-ratecan be determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSET™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, e.g., by combiningwith a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies for use in the methods described herein may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are reviewed, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., 2001) and further described, e.g., in the McCafferty et al.,Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Markset al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, inMethods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa,N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee etal., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad.Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g., a bispecific antibody is useful in a method describedherein. Multispecific antibodies are monoclonal antibodies that havebinding specificities for at least two different sites. In certainembodiments, one of the binding specificities is for one antigen (e.g.,CD79b) and the other is for any other antigen. In certain embodiments,one of the binding specificities is for one antigen (e.g., CD79b) andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofan antigen (e.g., CD79b). Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express the antigen (e.g.,CD79b). Bispecific antibodies can be prepared as full length antibodiesor antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebispecific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to CD79b as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a. Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b. Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout +3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c. Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Nonlimiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.,Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an anti-CD79b antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and S400 (EU numbering) of the heavy chain Fcregion. Cysteine engineered antibodies may be generated as described,e.g., in U.S. Pat. No. 7,521,541.

e. Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Nonlimiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

C. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an antibody described herein is provided.Such nucleic acid may encode an amino acid sequence comprising the VLand/or an amino acid sequence comprising the VH of the antibody (e.g.,the light and/or heavy chains of the antibody). In a further embodiment,one or more vectors (e.g., expression vectors) comprising such nucleicacid are provided. In a further embodiment, a host cell comprising suchnucleic acid is provided. In one such embodiment, a host cell comprises(e.g., has been transformed with): (1) a vector comprising a nucleicacid that encodes an amino acid sequence comprising the VL of theantibody and an amino acid sequence comprising the VH of the antibody,or (2) a first vector comprising a nucleic acid that encodes an aminoacid sequence comprising the VL of the antibody and a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VH of the antibody. In one embodiment, the host cell is eukaryotic,e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0,NS0, Sp20 cell). In one embodiment, a method of making an antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an antibody, nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

D. Assays

Antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to the target antigen. In certain embodiments, such a competingantibody binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by an antibody described herein. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized antigen is incubated in asolution comprising a first labeled antibody that binds to antigen(e.g., any of the antibodies described herein) and a second unlabeledantibody that is being tested for its ability to compete with the firstantibody for binding to antigen. The second antibody may be present in ahybridoma supernatant. As a control, immobilized antigen is incubated ina solution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to antigen, excess unbound antibody isremoved, and the amount of label associated with immobilized antigen ismeasured. If the amount of label associated with immobilized antigen issubstantially reduced in the test sample relative to the control sample,then that indicates that the second antibody is competing with the firstantibody for binding to antigen. See Harlow and Lane (1988) Antibodies:A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

E. Immunoconjugates

Provided herein are also immunoconjugates comprising an anti-CD79bantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes(i.e., a radioconjugate) for use in the methods described herein.

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Charm, R. V. (2008) Acc. Chem. Res. 41:98-107.

The ADC compounds of the invention include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase. Exemplary drug moieties include, but are notlimited to, a maytansinoid, dolastatin, auristatin, calicheamicin,anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene,CC1065, camptothecin, elinafide, and stereoisomers, isosteres, analogs,and derivatives thereof that have cytotoxic activity. Nonlimitingexamples of such immunoconjugates are discussed in further detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine. In some embodimentsof any of the methods, the immunoconjugate has the formula Ab-(L-D)p,wherein: (a) Ab is the antibody which binds a MM cell surface protein;(b) L is a linker; (c) D is a cytotoxic agent; and (d) p ranges from1-8.

An exemplary ADC has Formula I:

Ab-(L-D)_(p)  I

wherein p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:

-A_(a)-W_(w)—Y_(y)—  II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2; and Ab, D, and p are defined as above forFormula I. Exemplary embodiments of such linkers are described in U.S.Pat. No. 7,498,298, which is expressly incorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”.In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In some embodiments, a linker component comprises a “spacer” unit thatlinks the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit isp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or -cyno;m is an integer ranging from 0 to 4; and p ranges from 1 to about 20. Insome embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

Further nonlimiting exemplary ADCs include the structures:

wherein X is:

Y is:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃—) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I. Insome such embodiments, the antibody comprises more than one (linkerportion)^(a) substituents, such that more than one drug is coupled tothe antibody in the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents:bis-maleimido-trioxyethyletne glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitotic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al (2002) PNAS 99:7968-7973). Maytansinoids may also beprepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to,those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat.No. 4,256,746) (prepared, for example, by lithium aluminum hydridereduction of ansamytocin P2); C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared,for example, by acylation using acyl chlorides), and those havingmodifications at other positions of the aromatic ring.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, forexample, by the reaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

-   -   wherein the wavy line indicates the covalent attachment of the        sulfur atom of the maytansinoid drug moiety to a linker of an        ADC. Each R may independently be H or a C₁-C₆ alkyl. The        alkylene chain attaching the amide group to the sulfur atom may        be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S.        Pat. Nos. 633,410; 5,208,020; Chari et al (1992) Cancer Res.        52:127-131; Liu et al (1996) Proc. Natl. Acad. Sci USA        93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC of the invention, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. Nos. 7,276,497; 6,913,748; 6,441,163;633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al (2006) J.Med. Chem. 49:4392-4408, which are incorporated by reference in theirentirety). In some embodiments, the maytansinoid drug moiety has thefollowing stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

-   -   wherein the wavy line indicates the covalent attachment of the        sulfur atom of the drug to a linker (L) of an antibody-drug        conjugate.

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations (wherein Ab is antibody and p is 1 to about20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is1 to 4):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

-   -   wherein Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20.        In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or        p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1, the disclosures of which are hereby expressly incorporatedby reference. See also Liu et al. Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al. Cancer Research 52:127-131 (1992).

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (thedisclosure of which is hereby expressly incorporated by reference). Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Exemplary linking groups for making antibody-maytansinoid conjugatesinclude, for example, those described herein and those disclosed in U.S.Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari et al. Cancer Research52:127-131 (1992); US 2005/0276812 A1; and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.

(2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237;6,124,431). Auristatins are derivatives of the marine mollusk compounddolastatin-10. While not intending to be bound by any particular theory,dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin/auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172; Doronina et al (2003) NatureBiotechnology 21(7):778-784; Francisco et al (2003) Blood102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in U.S. Pat.Nos. 7,498,298 and 7,659,241, the disclosures of which are expresslyincorporated by reference in their entirety:

-   -   wherein the wavy line of DE and DF indicates the covalent        attachment site to an antibody or antibody-linker component, and        independently at each location:    -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R⁵ is independently H, COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R′⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R′)₂—C(R′)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R′ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R′ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹¹)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula DE is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula DF is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADC of Formula I comprising MMAE orMMAF and various linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.(2006) Bioconjugate Chem. 17:114-124). In some such embodiments, drugrelease is believed to be effected by antibody degradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schroder and K. Libke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. Nos. 7,498,298;5,635,483; 5,780,588; Pettit et al (1989) J. Am. Chem. Soc.111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277;Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J.Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In some embodiments, auristatin/dolastatin drug moieties of formulas DEsuch as MMAE, and DF, such as MMAF, and drug-linker intermediates andderivatives thereof, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, andMC-vc-PAB-MMAE, may be prepared using methods described in U.S. Pat. No.7,498,298; Doronina et al. (2006) Bioconjugate Chem. 17:114-124; andDoronina et al. (2003) Nat. Biotech. 21:778-784 and then conjugated toan antibody of interest.

(3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics, and analogues thereof, are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations (Hinman etal., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) CancerResearch 58:2925-2928). Calicheamicin has intracellular sites of actionbut, in certain instances, does not readily cross the plasma membrane.Therefore, cellular uptake of these agents through antibody-mediatedinternalization may, in some embodiments, greatly enhances theircytotoxic effects. Nonlimiting exemplary methods of preparingantibody-drug conjugates with a calicheamicin drug moiety are described,for example, in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; and5,767,285.

(4) Other Drug Moieties

Drug moieties also include geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof,including, but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or I¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al (1978)Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporateiodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRCPress 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananti-cancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asD-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; D-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature 312:604-608 (1984).

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g., p >5,may cause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g., hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

d) Certain Methods of Preparing Immunoconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g., lysine,(iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxylor amino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g., with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g.,by borohydride reagents to form stable amine linkages. In oneembodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either galactose oxidase or sodium meta-periodate mayyield carbonyl (aldehyde and ketone) groups in the antibody that canreact with appropriate groups on the drug (Hermanson, BioconjugateTechniques). In another embodiment, antibodies containing N-terminalserine or threonine residues can react with sodium meta-periodate,resulting in production of an aldehyde in place of the first amino acid(Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;

-   -   U.S. Pat. No. 5,362,852). Such an aldehyde can be reacted with a        drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

F. Methods and Compositions for Diagnostics and Detection

Provided herein are also methods and compositions for diagnosis and/ordetection of CD79b antibodies for use in the methods described hereinincluding detecting the presence of CD79b in a biological sample for usein selecting patients for treating using the methods described herein.The term “detecting” as used herein encompasses quantitative orqualitative detection. A “biological sample” comprises, e.g., a cell ortissue.

In one embodiment, an anti-CD79b antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of CD79b in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-CD79b antibody as described herein under conditionspermissive for binding of the anti-CD79b antibody to CD79b, anddetecting whether a complex is formed between the anti-CD79b antibodyand CD79b in the biological sample. Such method may be an in vitro or invivo method. In one embodiment, an anti-CD79b antibody is used to selectsubjects eligible for therapy with an anti-CD79b antibody, e.g., whereCD79b is a biomarker for selection of patients. In a further embodiment,the biological sample is a cell and/or tissue (e.g., bone marrow and/orblood).

In a further embodiment, an anti-CD79b antibody is used in vivo todetect, e.g., by in vivo imaging, a CD79b-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting a CD79b-positive cancer in a subject, the methodcomprising administering a labeled anti-CD79b antibody to a subjecthaving or suspected of having a CD79b-positive cancer, and detecting thelabeled anti-CD79b antibody in the subject, wherein detection of thelabeled anti-CD79b antibody indicates a CD79b-positive cancer in thesubject. In certain of such embodiments, the labeled anti-CD79b antibodycomprises an anti-CD79b antibody conjugated to a positron emitter, suchas ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ²⁴I. In a particularembodiment, the positron emitter is ⁸⁹Zr.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-CD79b antibody immobilized to a substrate with abiological sample to be tested for the presence of CD79b, exposing thesubstrate to a second anti-CD79b antibody, and detecting whether thesecond anti-CD79b is bound to a complex between the first anti-CD79bantibody and CD79b in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell or tissue (e.g., blood and/or bone marrow). In certainembodiments, the first or second anti-CD79b antibody is any of theantibodies described herein.

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include CD79b-positive cancers, such aslymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), Burkitt's lymphoma, diffuse B-cell lymphoma (DBCL), andmantle cell lymphoma, in particular, NHL, follicular lymphoma, and/orDBCL. In some embodiments, a CD79b-positive cancer is a cancer thatexpresses CD79b according to a reverse-transcriptase PCR (RT-PCR) assaythat detects CD79b mRNA. In some embodiments, the RT-PCR is quantitativeRT-PCR.

In certain embodiments, labeled anti-CD79b antibodies for use in themethods described herein are provided. Labels include, but are notlimited to, labels or moieties that are detected directly (such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels), as well as moieties, such as enzymes or ligands,that are detected indirectly, e.g., through an enzymatic reaction ormolecular interaction. Exemplary labels include, but are not limited to,the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, D-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like. In another embodiment, a label is apositron emitter. Positron emitters include but are not limited to ⁶⁸Ga,¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particular embodiment, apositron emitter is ⁸⁹Zr.

G. Pharmaceutical Formulations

Pharmaceutical formulations of any of the agents described herein (e.g.,anti-CD79b immunoconjugates) for use in any of the methods as describedherein are prepared by mixing such antibody or immunoconjugate havingthe desired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thedisorder and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or immunoconjugate of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or immunoconjugate; and(b) a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution or dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1—Anti-CD79b Immunoconjugate in Combination with Anti-CD20Antibody Plus Alkyating Agent (Bendamustine) in Lymphoma

The combination efficacy of anti-CD79b immunoconjugate (anti-CD79b(huMA79b.v28)-MC-vc-PAB-MMAE ADC; polatuzumab vedotin; Pola; DCDS4501A)with anti-CD20 antibody (rituximab) and bendamustine was evaluated in atumor xenograft model of WSU-DLCL2 human diffuse large B-cell lymphoma.

Female C.B-17 SCID mice (11-12 weeks old from Charles RiverLaboratories; Hollister, Calif.) were each inoculated subcutaneously inthe flank with 20 million WSU-DLCL2 cells (DSMZ, German Collection ofMicroorganisms an Cell Cultures; Braunschweig, Germany). When thexenograft tumors reached desired volume, animals were randomized intogroups of 9 mice each and received a single dose of treatments (referredto as Day 0). Anti-CD20 antibody (rituximab) was given intraperitoneallyat 30 mg/kg. Anti-CD79b-MMAE ADC and bendamustine was givenintravenously at 2 and 30 mg/kg, respectively.

Tumors were measured 1-2 times a week throughout the study usingUltraCal-IV calipers and tumor volume was calculated using followingformula: tumor volume (mm³)=0.5a×b², wherein a and b are the long andshort diameters of the tumor, respectively.

To appropriately analyze the repeated measurement of tumor volumes fromthe same animals over time, a mixed modeling approach was used (PinheiroJ, et al. nlme: linear and nonlinear mixed effects models. 2009; Rpackage, version 3.1-96). This approach addressed both repeatedmeasurements and modest dropout rates due to non-treatment relatedremoval of animals before the study end. Cubic regression splines wereused to fit a non-linear profile to the time courses of log 2 tumorvolume at each dose level. These non-linear profiles were then relatedto dose within the mixed model. The results were plotted as fitted tumorvolume of each group over time.

In this study, as shown in FIG. 1, anti-CD79b-MMAE ADC demonstratedclear inhibition of tumor growth, and the anti-tumor activity wascomparable with the combination of rituximab and bendamustine at thedoses tested. Additionally, the triple combination of anti-CD79b-MMAEADC with rituximab and bendamustine resulted in significantly greaterefficacy than the ADC or rituximab/bendamustine doublet alone.

Example 2—a Study of CD79b-MC-Vc-PAB-MMAE in Combination with Anti-CD20Antibody (Rituximab or Obinutuzumab) Plus Alkylating Agent(Bendamustine) in Patients with Relapsed or Refractory Follicular orDiffuse Large B-Cell Lymphoma

A multicenter, open-label study of polatuzumab vedotin (anti-CD79b(huMA79b.v28)-MC-vc-PAB-MMAE; “Pola”) administered by intravenous (IV)infusion in combination with standard doses of bendamustine (B) andrituximab (R) or obinutuzumab (G) in patients with relapsed orrefractory follicular lymphoma (FL) or diffuse large B-cell lymphoma(DLBCL) is initiated. The study comprises first stage dose-escalationstage, stage 2, and stage 3, and the time on treatment is 18-24 weeks.

In the first stage, FL and DLBCL patients are enrolled into separatecohorts for dose escalation of Pola in combination with R and B or G andB. Pola is administered intravenously on Day 2 of Cycle 1, then on Day 1of each subsequent cycle. R is administered at a dose of 375 mg/m²intravenously on Day 1 of Cycle 1 and on Day 1 of each subsequent cyclefor up to six cycles. B is administered intravenously (90 mg/m²) on Days2 and 3 of Cycle 1, then on Days 1 and 2 of each subsequent cycle. G isadministered intravenously (1000 mg) on Days 1, 8, and 15 of Cycle 1 andon Day 1 of each subsequent cycle for up to six cycles. Completeresponse (CR) rate is measured by positron emission tomography (PET)scan and is determined by an Institutional Review Board.

In the second stage, randomized, separate FL and DLBCL cohorts receive(a) Pola in combination with R and B or (b) R and B alone. R isadministered at a dose of 375 mg/m² intravenously on Day 1 of Cycle 1and on Day 1 of each subsequent cycle for up to six cycles. B isadministered intravenously (90 mg/m²) on Days 2 and 3 of Cycle 1, thenon Days 1 and 2 of each subsequent cycle.

In the third stage, non-randomized, separate FL and DLBCL cohortsreceive Pola in combination with G and B. B is administeredintravenously (90 mg/m²) on Days 2 and 3 of Cycle 1, then on Days 1 and2 of each subsequent cycle. G is administered intravenously (1000 mg) onDays 1, 8, and 15 of Cycle 1 and on Day 1 of each subsequent cycle forup to six cycles. Complete response (CR) rate is measured by positronemission tomography (PET) scan and is determined by an InstitutionalReview Board.

Inclusion criteria for patients on study includes:

-   -   Histologically confirmed FL (Grade 1, 2, or 3a) or DLBCL    -   Must have received at least one prior therapy for FL or DLBCL.        Patients must have either relapsed or have become refractory to        a prior regimen as defined below:

(a) Relapsed/Refractory FL: Patients who have relapsed to priorregimen(s) after having a documented history of response (completeresponse [CR], CR unconfirmed [CRu], or partial response [PR]) of >/=6months in duration from completion of regimen(s); refractory to anyprior regimen, defined as no response to the prior therapy, orprogression within 6 months of completion of the last dose of therapy.

(b) Relapsed/Refractory DLBCL: Patients who are ineligible forsecond-line stem cell transplant (SCT), with progressive disease or noresponse (stable disease [SD])<6 months from start of initial therapy;patients who are ineligible for second-line SCT, with disease relapseafter initial response of >/=6 months from start of initial therapy;patients who are ineligible for third-line (or beyond) SCT, withprogressive disease or no response (SD)<6 months from start of priortherapy; patients who are ineligible for third-line (or beyond) SCT withdisease relapse after initial response of >/=6 months from start ofprior therapy.

-   -   If the patient has received prior bendamustine, response        duration must have been >1 year (for patients who have relapse        disease after a prior regimen).    -   At least one bi-dimensionally measurable lesion on imaging scan        defined as >1.5 cm in its longest dimension; confirmed        availability of archival or freshly collected tumor tissue        meeting protocol-defined specifications prior to study        enrollment; Life expectancy of at least 24 weeks; Eastern        Cooperative Oncology Group (ECOG) Performance Status of 0, 1, or        2; adequate hematological function; and, for women of        childbearing potential, a negative serum pregnancy test result        within 7 days prior to commencement of dosing.

Exclusion criteria for patients on study includes: history of severeallergic or anaphylactic reactions to humanized or murine monoclonalantibodies (MAbs, or recombinant antibody-related fusion proteins) orknown sensitivity or allergy to murine products, contraindication tobendamustine, rituximab, or obinutuzumab, history of sensitivity tomannitol, prior use of any MAb, radioimmunoconjugate, or antibody-drugconjugate (ADC) within 4 weeks before Cycle 1 Day 1, treatment withradiotherapy, chemotherapy, immunotherapy, immunosuppressive therapy, orany investigational agent for the purposes of treating cancer within 2weeks prior to Cycle 1 Day 1, ongoing corticosteroid use >30 mg/dayprednisone or equivalent, for purposes other than lymphoma symptomcontrol, completion of autologous SCT within 100 days prior to Cycle 1Day 1, prior allogeneic SCT, eligibility for autologous SCT (patientswith relapsed/refractory DLBCL), Grade 3b FL, history of transformationof indolent disease to DLBCL, primary CNS lymphoma, current Grade >1peripheral neuropathy, evidence of significant, uncontrolled concomitantdiseases that could affect compliance with the protocol orinterpretation of results, including significant cardiovascular disease(such as New York Heart Association Class III or IV cardiac disease,myocardial infarction within the last 6 months, unstable arrhythmias, orunstable angina) or significant pulmonary disease (including obstructivepulmonary disease and history of bronchospasm), known active bacterial,viral, fungal, mycobacterial, parasitic, or other infection (excludingfungal infections of nail beds) at study enrollment or any major episodeof infection requiring treatment with intravenous (IV) antibiotics orhospitalization within 4 weeks prior to Cycle 1 Day 1, patients withsuspected or latent tuberculosis, positive test results for chronichepatitis B virus (HBV) infection or for hepatitis C virus (HCV)antibody, known infection with HIV or human T-cell leukemia virus 1(HTLV-1) virus, women who are pregnant or lactating or who intend tobecome pregnant within a year of the last dose of rituximab orobinutuzumab, evidence of laboratory abnormalities in standard renal,hepatic or coagulation function tests.

Example 3—Anti-CD79b Immunoconjugate in Combination with Bcl2 Inhibitorin Lymphoma

The combination efficacy of anti-CD79b-MMAE ADC (DCDS4501A) with aselective Bcl2 inhibitor (ABT-199 (i.e., venetoclax, GDC-0199,4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide,and/or CAS #1257044-40-8) was evaluated in a tumor xenograft model ofGranta-519 human mantle-cell lymphoma.

Female C.B-17 SCID mice (8 weeks old from Charles River Laboratories;Hollister, Calif.) were each inoculated in the flank with 20 millionGranta-519 cells. When the xenograft tumors reached desired volume,animals were randomized into groups of 9 mice each and receivedtreatments (Day 0 of the study). Anti-CD79b-MC-vc-PAB-MMAE ADC was dosedonce intravenously at 1 mg/kg and ABT-199 were given orally once a dayfor 21 days at 100 mg/kg.

Tumors were measured 1-2 times a week throughout the study usingUltraCal-IV calipers and tumor volume was calculated using followingformula: tumor volume (mm³)=0.5a×b², wherein a and b are the long andshort diameters of the tumor, respectively.

To appropriately analyze the repeated measurement of tumor volumes fromthe same animals over time, a mixed modeling approach was used (PinheiroJ, et al. nlme: linear and nonlinear mixed effects models. 2009; Rpackage, version 3.1-96). This approach addresses both repeatedmeasurements and modest dropout rates due to non-treatment relatedremoval of animals before the study end. Cubic regression splines wereused to fit a non-linear profile to the time courses of log 2 tumorvolume at each dose level. These non-linear profiles were then relatedto dose within the mixed model. The results were plotted as fitted tumorvolume of each group over time.

In this study, as shown in FIG. 2, treatment withanti-CD79b-MC-vc-PAB-MMAE ADC alone caused modest tumor growth delaywhile ABT-199 monotherapy did not result in anti-tumor activity.However, the combination of anti-CD79b-MC-vc-PAB-MMAE ADC and ABT-199resulted in greater efficacy, causing tumor regressions, than eitheragent alone. The combination of anti-CD79b-MC-vc-PAB-MMAE ADC andABT-199 was well-tolerated based on minimal changes in animal bodyweights during the treatment period.

Example 4—Anti-CD79b Immunoconjugate in Combination Therapy in Lymphoma

The combination efficacy of anti-CD79b-MC-vc-PAB-MMAE ADC (DCDS4501A;huMA79bv28-MC-vc-PAB-MMAE) with various combination therapies wasevaluated in a tumor xenograft model of WSU-DLCL2 (DLBCL).

Female C.B-17 SCID mice (14 weeks old from Charles River Laboratories;Hollister, Calif.) were each inoculated in the flank with 20 millionWSU-DLCL2 (DLBCL) cells. When the xenograft tumors reached desiredvolume, animals were randomized and received treatments (Day 0 of thestudy). There were six treatment groups (1) vehicle, (2)anti-CD79b-vcMMAE, (3) G-CHP (GA101-cyclophosphamide, doxorubicin, andprednisone), (4) G-bendamustine (GA101-bendamustine), (5) G-CHP(GA101-cyclophosphamide, doxorubicin, andprednisone)+anti-CD79b-MC-vc-PAB-MMAE, and (6) G-bendamustine(GA101-bendamustine)+anti-CD79b-MC-vc-PAB-MMAE.

CD79b-MC-vc-PAB-MMAE ADC was dosed once intravenously at 2 mg/kg, iv,once. GA101 dosed 30 mg/kg, ip, once. CHP was dosed cyclophosphamide, 30mg/kg, iv, once+doxorubicin, 2.475 mg/kg, iv, once+prednisone, 0.15mg/kg, po, qdx5. Bendamustine was dosed 30 mg/kg, iv, once.

As described above, tumors were measured 1-2 times a week throughout thestudy using UltraCal-IV calipers and tumor volume was calculated usingfollowing formula: tumor volume (mm³) -0.5a×b², wherein a and b are thelong and short diameters of the tumor, respectively.

In this study, as shown in FIG. 3A, treatment withanti-CD79b-MC-vc-PAB-MMAE ADC combined well with G-CHP (or G-Benda) withbetter efficacy than the anti-CD79b-MC-vc-PAB-MMAE ADC or G-CHP (orG-Benda) alone. The combinations were well-tolerated based on minimalchanges in animal body weights during the treatment period.

Example 5—Anti-CD79b Immunoconjugate in Combination Therapy in Lymphoma

The combination efficacy of anti-CD79b-MC-vc-PAB-MMAE ADC (DCDS4501A;huMA79bv28-MC-vc-PAB-MMAE) with various combination therapies wasevaluated in a tumor xenograft model of TMD8 (ABC-DLBCL).

Female C.B-17 SCID mice (13 weeks old from Charles River Laboratories;Hollister, Calif.) were each inoculated in the flank with 5 million TMD8(ABC-DLBCL) cells. When the xenograft tumors reached desired volume,animals were randomized and received treatments (Day 0 of the study).There were seven treatment groups (1) vehicle, (2) GA101, (3)anti-CD79b-MC-vc-PAB-MMAE, (4) lenalidomide, (5)GA101+anti-CD79b-MC-vc-PAB-MMAE, (6) GA101+lenalidomide, and (7)GA101+lenalidomide+anti-CD79b-MC-vc-PAB-MMAE. CD79b-MC-vc-PAB-MMAE ADCwas dosed once intravenously at 2 mg/kg, iv, once. GA101 dosed 1 mg/kg,ip, qwx3. Lenalidomide was administered at 20 mg/kg, po, (qdx5)x3.

As described above, tumors were measured 1-2 times a week throughout thestudy using UltraCal-IV calipers and tumor volume was calculated usingfollowing formula: tumor volume (mm³) -0.5a×b², wherein a and b are thelong and short diameters of the tumor, respectively.

Literature (Br J Haematol 2013 Zhang et al.) reported that lenalidomidepreferentially suppresses the growth of the activated B-cell-like (ABC)subtype, with minimal effect on non-ABC-DLBCL cells. In this study usingABC-DLBCL, as shown in FIG. 3B, treatment with lenalidomide monotherapyshowed little efficacy in this model. Further, combining lenalidomidewith GA101 did not provide additional efficacy over GA101 alone.However, use of anti-CD79b-MC-vc-PAB-MMAE ADC alone or in combination inthe ABC subtype showed strong efficacy with tumor regression. Inaddition, all treatments were well tolerated.

Example 6—Anti-CD79b Immunoconjugate in Combination Therapy in Lymphoma

The combination efficacy of anti-CD79b-MC-vc-PAB-MMAE ADC (DCDS4501A;huMA79bv28-MC-vc-PAB-MMAE) with various combination therapies wasevaluated in a tumor xenograft model of WSU-DLCL2 (DLBCL).

Female C.B-17 SCID mice (13 weeks old from Charles River Laboratories;Hollister, Calif.) were each inoculated in the flank with 20 millionWSU-DLCL2 (DLBCL) cells. When the xenograft tumors reached desiredvolume, animals were randomized and received treatments (Day 0 of thestudy). There were twelve treatment groups (1) vehicle, (2) GA101, (3)Bcl2i (GDC-199), (4) PI3Ki (GDC-032), (5) anti-CD79b-MC-vc-PAB-MMAE ADC,(6) GA101+anti-CD79b-MC-vc-PAB-MMAE, (7) GA101+Bcl2i (GDC-199), (8)GA101+PI3Ki (GDC-032), (9) GA101+Bcl2i(GDC-199)+anti-CD79b-MC-vc-PAB-MMAE, (10) GA101+PI3Ki(GDC-032)+anti-CD79b-MC-vc-PAB-MMAE, (11) Rituximab, and (12)Rituximab+anti-CD79b-MC-vc-PAB-MMAE.

CD79b-MC-vc-PAB-MMAE ADC was dosed once intravenously at 2 mg/kg, iv,once. GA101 dosed 30 mg/kg, ip, once. Bcl2 inhibitor, GDC-199, was dosedat 100 mg/kg, po, qdx21. PI3K inhibitor, GDC-032 was dosed at 10 mg/kg,po, qdx21. Rituximab was dosed at 30 mg/kg, ip, once.

As described above, tumors were measured 1-2 times a week throughout thestudy using UltraCal-IV calipers and tumor volume was calculated usingfollowing formula: tumor volume (mm³) -0.5a×b², wherein a and b are thelong and short diameters of the tumor, respectively. Results are shownin FIG. 4. In this study, the Bcl2 inhibitor, GDC-0199, PI3K inhibitor,GDC-0032, and anti-CD20 (GA101 or Rituximab) monotherapy at the dosestested had little effect on the tumor growth. However, efficacy becamemore apparent when combining Bcl2 inhibitor GDC-0199 with anti-CD20,GA101. Furthermore, among all the different treatments evaluated thetriple combination of anti-CD79b-vcMMAE, GA101 and Bcl2 inhibitordisplayed the greatest efficacy, causing complete tumor remission.

Example 7—anti-CD79b Immunoconjugate in Combination with Venetoclax

This study will evaluate the efficacy, safety, and pharmacokinetics ofthe combination of obinutuzumab (GA101 or G) plus polatuzumab vedotin(anti-CD79b(huMA79b.v23)-MC-vc-PAB-MMAE ADC (DCDS4501A) or pola) plus aselective Bcl2 inhibitor (ABT-199 (i.e., venetoclax, GDC-0199,4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide,V, and/or CAS #1257044-40-8) (G+pola+V) in patients with relapse orrefractory (R/R) follicular lymphoma (FL) or diffuse large B celllymphoma (DLBCL).

Efficacy Objectives: Response will be determined on the basis ofpositron emission tomography and computed tomography (PET-CT) scans orCT scans alone, using Revised Lugano Response Criteria for MalignantLymphoma, hereinafter referred to as Lugano 2014 criteria. Response willbe determined by an Independent Review Committee (IRC) and by theinvestigator. The primary efficacy objective for this study is toevaluate the efficacy of G+Pola+V on the basis of the followingendpoint: Complete response (CR) at end of induction (EOI), asdetermined by the IRC on the basis of PET-CT scans.

The secondary efficacy objective for this study is to evaluate theefficacy of G+Pola+V on the basis of the following endpoints: CR at EOI,as determined by the investigator on the basis of PET-CT scans, CR atEOI, as determined by the investigator on the basis of CT scans alone,Objective response (defined as a CR or partial response [PR]) at EOI, asdetermined by the IRC and by the investigator on the basis of PET-CTscans, Objective response (defined as a CR or PR) at EOI, as determinedby the IRC and by the investigator on the basis of CT scans alone, Bestresponse of CR or PR during the study, as determined by the investigatoron the basis of CT scans alone.

The exploratory efficacy objective for this study is to evaluate thelong-term efficacy of G+Pola+V on the basis of the following endpoints:for patients who have positive PET scans at EOJ: CR at 12 months, asdetermined by the IRC and by the investigator on the basis of PET-CTscans, progression-free survival, defined as the time from initiation ofstudy treatment to first occurrence of disease progression or relapse,as determined by investigator on the basis of CT scans alone, or deathfrom any cause, event-free survival, defined as the time from initiationof study treatment to any treatment failure, including diseaseprogression or relapse, as determined by investigator on the basis of CTscans alone, initiation of new anti-lymphoma therapy, or death from anycause, whichever occurs first, disease-free survival, defined, amongpatients who achieve a CR, as the time from the first occurrence of adocumented CR to relapse, as determined by the investigator on the basisof CT scans alone, or death from any cause, whichever occurs first,overall survival, and defined as the time from initiation of studytreatment to death from any cause.

All patients enrolled in the dose-escalation phase will receiveinduction treatment, administered in 21-day cycles. When studytreatments are given on the same day, they will be administeredsequentially in the following order: venetoclax, obinutuzumab, andpolatuzumab vedotin.

Cycle 1:

-   -   Venetoclax 400, 600, or 800 mg by mouth (PO) once daily on Days        1-21    -   Obinutuzumab 1000 mg IV on Days 1, 8, and 15    -   Polatuzumab vedotin 1.4 or 1.8 mg/kg intravenously (IV) on Day 1

Cycles 2-6:

-   -   Venetoclax 400, 600, or 800 mg PO once daily on Days 1-21    -   Obinutuzumab 1000 mg IV on Day 1    -   Polatuzumab vedotin 1.4 or 1.8 mg/kg IV on Day 1

After completion of induction treatment, patients with FL will continueto receive daily venetoclax treatment (during Month 1) until response isassessed at EOI. Venetoclax will be discontinued if response assessmentsat EOI indicate that a patient is not eligible for post-inductiontreatment (referred to as maintenance). Patients who achieve a CR, PR,or SD at EOI will receive maintenance treatment with obinutuzumab andvenetoclax. Polatuzumab vedotin will not be given as maintenancetreatment. Maintenance treatment will continue until disease progressionor unacceptable toxicity for up to 24 months. When study treatments aregiven on the same day, venetoclax will be administered prior toobinutuzumab.

Treatments will be administered as follows:

-   -   Venetoclax 400, 600, or 800 mg PO once daily for 8 months        (Months 1-8)    -   Obinutuzumab 1000 mg IV on Day 1 of every other month (i.e.,        every 2 months) for 24 months, starting with Month 2 (e.g.,        Months 2, 4, 6, 8, etc.).

A 3+3 dose-escalation schema will be used. The obinutuzumab dose willremain fixed at 1000 mg during the dose-escalation phase. The startingdoses in Cohort 1 are 1.4 mg/kg for polatuzumab vedotin and 400 mg forvenetoclax. In Cohorts 2-6, dose escalation of polatuzumab vedotin andvenetoclax will proceed in increments that parallel the magnitude ofdose increases tested in ongoing Phase Ib trials. For polatuzumabvedotin, there are 2 possible dose levels: 1.4 or 1.8 mg/kg. Forvenetoclax, there are 3 possible dose levels: 400, 600, or 800 mg.Intrapatient dose escalation is not allowed.

All patients enrolled in the expansion phase will receive inductiontreatment, administered in 21-day cycles. When study treatments aregiven on the same day, they will be administered sequentially in thefollowing order: venetoclax, obinutuzumab, and polatuzumab vedotin.

Cycle 1:

-   -   Venetoclax at the RP2D (mg) PO once daily on Days 1-21    -   Obinutuzumab 1000 mg IV on Days 1, 8, and 15    -   Polatuzumab vedotin at the RP2D (mg/kg) IV on Day 1

Cycles 2-6:

-   -   Venetoclax at the RP2D (mg) PO once daily on Days 1-21    -   Obinutuzumab 1000 mg IV on Day 1    -   Polatuzumab vedotin at the RP2D (mg/kg) IV on Day 1

After completion of induction treatment, patients will continue toreceive daily venetoclax treatment (during Month 1) until response isassessed at EOI. Venetoclax will be discontinued if response assessmentsat EOI indicate that a patient is not eligible for post-inductiontreatment. Patients with DLBCL who achieve a CR or PR at EOI willreceive post-induction treatment (referred to as consolidation) withobinutuzumab and venetoclax, and patients with FL who achieve a CR, PR,or SD at EOI will receive post-induction treatment (referred to asmaintenance) with obinutuzumab and venetoclax. Polatuzumab vedotin willnot be given as post-induction treatment. Post-induction treatment willcontinue until disease progression or unacceptable toxicity for up to 8months for consolidation treatment or 24 months for maintenancetreatment. When study treatments are given on the same day, venetoclaxwill be administered prior to obinutuzumab.

Diffuse Large B-Cell Lymphoma: Consolidation treatment consisting of thefollowing, administered for 8 months (Months 1-8):

-   -   Venetoclax at the RP2D (mg) PO once daily for 8 months (Months        1-8)    -   Obinutuzumab 1000 mg IV on Day 1 of every other month (i.e.,        every 2 months), starting with Month 2 (i.e., Months 2, 4, 6,        and 8)

Follicular Lymphoma: Maintenance treatment consisting of the following,administered for 24 months (Months 1-24):

-   -   Venetoclax at the RP2D (mg) PO once daily for 8 months (Months        1-8)    -   Obinutuzumab 1000 mg IV on Day 1 of every other month (i.e.,        every 2 months) for 24 months, starting with Month 2 (e.g.,        Months 2, 4, 6, 8, etc.).

Inclusion Criteria: Patients must meet the following criteria for studyentry: signed Informed Consent Form, age ≥18 years, Eastern CooperativeOncology Group Performance Status of 0, 1, or 2, for patients enrolledin the dose-escalation phase: R/R FL after treatment with at least 1prior chemoimmunotherapy regimen that included an anti-CD20 monoclonalantibody and for which no other more appropriate treatment optionexists, as determined by the investigator, for patients enrolled in theexpansion phase: B-cell lymphoma classified as either of the following:—R/R FL after treatment with at least 1 prior chemoimmunotherapy regimenthat included an anti-CD20 monoclonal antibody and for which no othermore appropriate treatment option exists, as determined by theinvestigator —R/R DLBCL after treatment with at least 1 priorchemoimmunotherapy regimen that included an anti-CD20 monoclonalantibody, with no curative option as determined by the investigator,histologically documented CD20-positive non-Hodgkin's lymphoma asdetermined by the local laboratory, fluorodeoxyglucose-avid lymphoma(i.e., PET-positive lymphoma), at least one bi-dimensionally measurablelesion (>1.5 cm in its largest dimension by CT scan or magneticresonance imaging), availability of a representative tumor specimen andthe corresponding pathology report for retrospective centralconfirmation of the diagnosis of FL or DLBCL. If the archival tissue isunavailable or unacceptable, a pretreatment core, excisional, orincisional tumor biopsy is required. Cytological or fine-needleaspiration samples are not acceptable. If the patient receivedanti-lymphoma treatment between the time of the most recent availablebiopsy and initiation of study treatment, a core-needle biopsy isstrongly recommended.

Exclusion Criteria: Patients who meet any of the following criteria willbe excluded from study entry: known CD20-negative status at relapse orprogression, prior allogeneic stem cell transplant (SCT), completion ofautologous SCT within 100 days prior to Day 1 of Cycle 1, prior standardor investigational anti-cancer therapy as specified:—Radioimmunoconjugate within 12 weeks prior to Day 1 of Cycle 1,—Monoclonal antibody or antibody-drug conjugate therapy within 4 weeksprior to Day 1 of Cycle 1, and —Radiotherapy, chemotherapy, hormonaltherapy, or targeted small-molecule therapy within 2 weeks prior to Day1 of Cycle 1, clinically significant toxicity (other than alopecia) fromprior therapy that has not resolved to Grade ≤2 (per NCI CTCAE v4.0)prior to Day 1 of Cycle 1, current Grade >1 peripheral neuropathy, •CNSlymphoma or leptomeningeal infiltration, treatment with systemiccorticosteroids >20 mg/day prednisone or equivalent, patients who arereceiving corticosteroids ≤20 mg/day prednisone or equivalent must bedocumented to be on a stable dose for at least 4 weeks prior to Day 1 ofCycle 1. If corticosteroid treatment is urgently required for lymphomasymptom control prior to the start of study treatment, up to 100 mg/dayof prednisone or equivalent can be given for a maximum of 5 days, butall tumor assessments must be completed prior to start of corticosteroidtreatment. History of severe allergic or anaphylactic reaction tohumanized or murine monoclonal antibodies known sensitivity or allergyto murine products or any component of the obinutuzumab, polatuzumabvedotin, or venetoclax formulations, active bacterial, viral, fungal, orother infection, caution should be exercised when considering the use ofobinutuzumab in patients with a history of recurring or chronicinfections, requirement for warfarin treatment (because of potentialdrug-drug interactions that may increase the exposure of warfarin),treatment with the following agents within 7 days prior to the firstdose of venetoclax: —Strong CYP3A inhibitors such as fluconazole,ketoconazole, and clarithromycin and —Strong CYP3A inducers such asrifampin and carbamazepine, consumption of grapefruit, grapefruitproducts, Seville oranges (including marmalade that contains Sevilleoranges), or star fruit within 3 days prior to the first dose ofvenetoclax, clinically significant history of liver disease, includingviral or other hepatitis, current alcohol abuse, or cirrhosis, positivefor hepatitis B surface antigen, total hepatitis B core antibody, orhepatitis C virus antibody at screening, known history of HIV positivestatus, for patients with unknown HIV status, HIV testing will beperformed at screening if required by local regulations, history ofprogressive multifocal leukoencephalopathy, vaccination with a livevirus vaccine within 28 days prior to Day 1 of Cycle 1, history of othermalignancy that could affect compliance with the protocol orinterpretation of results, with the exception of the following:—Curatively treated carcinoma in situ of the cervix, good-prognosisductal carcinoma in situ of the breast, basal- or squamous-cell skincancer, Stage I melanoma, or low-grade, early-stage localized prostatecancer —Any previously treated malignancy that has been in remissionwithout treatment for ≥2 years prior to enrollment, evidence of anysignificant, uncontrolled concomitant disease that could affectcompliance with the protocol or interpretation of results, includingsignificant cardiovascular disease (such as New York Heart AssociationClass III or IV cardiac disease, myocardial infarction within theprevious 6 months, unstable arrhythmia, or unstable angina) orsignificant pulmonary disease (such as obstructive pulmonary disease orhistory of bronchospasm), major surgical procedure other than fordiagnosis within 28 days prior to Day 1 of Cycle 1, or anticipation of amajor surgical procedure during the course of the study, inadequatehematologic function (unless due to underlying lymphoma), defined asfollows: —Hemoglobin <9 g/dL, —ANC <1.5×10⁹/L, and —Platelet count<75×10⁹/L.

Example 8—anti-CD79b Immunoconjugate in Combination with Lenalidomide

This study will evaluate the safety, efficacy, and pharmacokinetics ofinduction treatment consisting of obinutuzumab (GA101 or G) incombination with polatuzumab vedotin(anti-CD79b(huMA79b.v23)-MC-vc-PAB-MMAE ADC (DCDS4501A) or pola) andlenalidomide (Len) (G+Pola+Len) in patients with relapsed or refractoryfollicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL),followed by post induction treatment with obinutuzumab in combinationwith lenalidomide in patients with FL who achieve a complete response(CR), partial response (PR), or stable disease at end of induction (EOI)and in patients with DLBCL who achieve a CR or PR at EOI. Specificobjectives and corresponding endpoints for the study are outlined below.

Response will be determined on the basis of positron emission tomography(PET) and computed tomography (CT) scans or CT scans alone, usingRevised Lugano Response Criteria for Malignant Lymphoma, hereinafterreferred to as the Lugano 2014 criteria. Response will be determined byan Independent Review Committee (IRC) and by the investigator.

Primary Efficacy Objective: The primary efficacy objective for thisstudy is to evaluate the efficacy of induction treatment with G+Pola+Lenon the basis of the following endpoint: CR at EOI, as determined by theIRC on the basis of PET-CT scans.

Secondary Efficacy Objectives: The secondary efficacy objective for thisstudy is to evaluate the efficacy of induction treatment with G+Pola+Lenon the basis of the following endpoints: CR at EOI, as determined by theinvestigator on the basis of PET-CT scans, CR at EOI, as determined bythe investigator on the basis of CT scans alone, Objective response(defined as a CR or PR) at EOI, as determined by the IRC and by theinvestigator on the basis of PET-CT scans, Objective response (definedas a CR or PR) at EOI, as determined by the IRC and by the investigatoron the basis of CT scans alone, Best response of CR or PR during thestudy, as determined by the investigator on the basis of CT scans alone.

Exploratory Efficacy Objective: The exploratory efficacy objective forthis study is to evaluate the long-term efficacy of G+Pola+Len on thebasis of the following endpoints: for patients who have positive PETscans at EOJ: CR at 12 months, as determined by the IRC and by theinvestigator on the basis of PET-CT scans, PFS, defined as the time frominitiation of study treatment to first occurrence of disease progressionor relapse, as determined by investigator on the basis of CT scansalone, or death from any cause, EFS, defined as the time from initiationof study treatment to any treatment failure, including diseaseprogression or relapse, as determined by investigator on the basis of CTscans alone, initiation of new anti-lymphoma therapy, or death from anycause, whichever occurs first, disease-free survival, defined, amongpatients achieving a CR, as the time from the first occurrence of adocumented CR to relapse, as determined by the investigator on the basisof CT scans alone, or death from any cause, whichever occurs first, andoverall survival, defined as the time from initiation of study treatmentto death from any cause.

Inclusion Criteria: Patients must meet the following criteria for studyentry: signed Informed Consent Form, age ≥18 years, Eastern CooperativeOncology Group Performance Status of 0, 1, or 2. For patients enrolledin the dose-escalation phase: relapsed or refractory FL after treatmentwith at least one prior chemoimmunotherapy regimen that included ananti-CD20 monoclonal antibody and for which no other more appropriatetreatment option exists as determined by the investigator. For patientsenrolled in the expansion phase: lymphoma classified as either of thefollowing: relapsed or refractory FL after treatment with at least oneprior chemoimmunotherapy regimen that included an anti-CD20 monoclonalantibody and for which no other more appropriate treatment option existsas determined by the investigator, relapsed or refractory DLBCL aftertreatment with at least one prior chemoimmunotherapy regimen in patientswho are not eligible for autologous stem-cell transplantation or whohave experienced disease progression following treatment with high-dosechemotherapy plus autologous stem-cell transplantation, histologicallydocumented CD20-positive B-cell lymphoma as determined by the locallaboratory, fluorodeoxyglucose-avid lymphoma (i.e., PET-positivelymphoma), at least one bi-dimensionally measurable lesion (>1.5 cm inits largest dimension by CT scan or magnetic resonance imaging),availability of a representative tumor specimen and the correspondingpathology report for retrospective central confirmation of the diagnosisof FL or DLBCL. If the archival tissue is unavailable or unacceptable, apretreatment core-needle, excisional, or incisional tumor biopsy isrequired. Cytological or fine-needle aspiration samples are notacceptable.

Exclusion Criteria: Patients who meet any of the following criteria willbe excluded from study entry: known CD20-negative status at relapse orprogression, central nervous system lymphoma or leptomeningealinfiltration, prior allogeneic stem-cell transplantation (SCT),completion of autologous SCT within 100 days prior to Day 1 of Cycle 1,history of resistance to lenalidomide or response duration of <1 year(for patients who had a response to a prior lenalidomide-containingregimen), prior standard or investigational anti-cancer therapy asspecified: Lenalidomide, fludarabine, or alemtuzumab within 12 monthsprior to Day 1 of Cycle1, radioimmunoconjugate within 12 weeks prior toDay 1 of Cycle 1, monoclonal antibody or antibody-drug conjugate therapywithin 4 weeks prior to Day 1 of Cycle 1, radiotherapy, chemotherapy,hormonal therapy, or targeted small-molecule therapy within 2 weeksprior to Day 1 of Cycle 1, clinically significant toxicity (other thanalopecia) from prior therapy that has not resolved to Grade ≤2 (per NCICTCAE, Version 4.0) prior to Day 1 of Cycle 1, treatment with systemicimmunosuppressive medications, including, but not limited to,prednisone, azathioprine, methotrexate, thalidomide, and anti-tumornecrosis factor agents within 2 weeks prior to Day 1 of Cycle 1.Treatment with inhaled corticosteroids and mineralocorticoids ispermitted, if corticosteroid treatment is urgently required for lymphomasymptom control prior to the start of study treatment, up to 100 mg/dayof prednisone or equivalent can be given for a maximum of 5 days, butall tumor assessments must be completed prior to initiation ofcorticosteroid treatment, history of severe allergic or anaphylacticreaction to humanized or murine monoclonal antibodies, known sensitivityor allergy to murine products or any component of obinutuzumab,polatuzumab vedotin, or lenalidomide formulations, history of erythemamultiforme, Grade ≥3 rash, or desquamation (blistering) following priortreatment with immunomodulatory derivatives such as thalidomide andlenalidomide, active bacterial, viral, fungal, or other infection,caution should be exercised when considering the use of obinutuzumab inpatients with a history of recurring or chronic infections, positive forhepatitis B surface antigen, total hepatitis B core antibody, orhepatitis C virus antibody at screening, known history of HIV positivestatus, history of progressive multifocal leukoencephalopathy,vaccination with a live virus vaccine within 28 days prior to Day 1 ofCycle 1, history of other malignancy that could affect compliance withthe protocol or interpretation of results, with the exception of thefollowing: curatively treated carcinoma in situ of the cervix;good-prognosis ductal carcinoma in situ of the breast; basal- orsquamous-cell skin cancer; Stage I melanoma; or low-grade, early-stagelocalized prostate cancer, any previously treated malignancy that hasbeen in remission without treatment for ≥2 years prior to enrollment,contraindication to treatment for TE prophylaxis, Grade ≥2 neuropathy,evidence of any significant, uncontrolled concomitant disease that couldaffect compliance with the protocol or interpretation of results,including significant cardiovascular disease (such as New York HeartAssociation Class III or IV cardiac disease, myocardial infarctionwithin the previous 6 months, unstable arrhythmia, or unstable angina)or significant pulmonary disease (such as obstructive pulmonary diseaseor history of bronchospasm), major surgical procedure other than fordiagnosis within 28 days prior to Day 1 of Cycle 1 or anticipation of amajor surgical procedure during the course of the study, inadequatehematologic function (unless due to underlying lymphoma), defined asfollows: Hemoglobin <9 g/dL, ANC <1.5×10⁹/L, Platelet count <75×10⁹/L,any of the following abnormal laboratory values (unless due tounderlying lymphoma): calculated creatinine clearance <60 mL/min (usingthe Cockcroft-Gault formula), AST or ALT >2.5× upper limit of normal(ULN), serum total bilirubin >1.5×ULN (or >3×ULN for patients withGilbert syndrome), INR or PT >1.5×ULN in the absence of therapeuticanticoagulation, or PTT or aPTT >1.5×ULN in the absence of a lupusanticoagulant.

Obinutuzumab: Induction-Patients will receive obinutuzumab 1000 mgintravenously on Days 1, 8, and 15 of Cycle 1 and on Day 1 of eachsubsequent 28-day cycle for up to 6 cycles. Post-Induction—Forconsolidation treatment, patients with DLBCL will receive obinutuzumab1000 mg intravenously on Day 1 of every other month for approximately 6months of additional treatment. For maintenance treatment, patients withFL will receive obinutuzumab 1000 mg intravenously on Day 1 of everyother month for approximately 24 months of additional treatment.

Polatuzumab Vedotin: Induction-Patients will receive polatuzumab vedotin1.4 or 1.8 mg/kg intravenously on Day 1 of each 28-day cycle for up to 6cycles. •In the Phase Ib portion of the study, the total dose ofpolatuzumab vedotin for each patient will depend on dose-levelassignment and the patient's weight on Day 1 of Cycle 1 (or within 96hours before Day 1 of Cycle 1). In the Phase II portion of the study,the total dose of polatuzumab vedotin for each patient will depend onthe RP2D established in the Phase Ib portion and the patient's weight onDay 1 of Cycle 1 (or within 96 hours before Day 1 of Cycle 1).Post-Induction-No polatuzumab vedotin will be administered.

Lenalidomide: Induction-Patients will receive lenalidomide 10, 15, or 20mg orally once daily on Days 1-21 of each 28-day cycle for up to 6cycles. In the Phase Ib portion of the study, the dose of lenalidomidefor each patient will depend on dose-level assignment on Day 1 ofCycle 1. In the Phase II portion of the study, the dose of lenalidomidefor each patient will depend on the RP2D established in the Phase Ibportion of the study. Post-Induction-Patients will receive lenalidomide10 mg orally once daily on Days 1-21 of each month. For consolidationtreatment for patients with DLBCL will receive lenalidomide 10 mg orallyonce daily on Days 1-21 of each month (starting Month 1 and continuingthrough Month 6) for approximately 6 months of additional treatment. Formaintenance treatment, patients with FL will receive lenalidomide 10 mgorally once daily on Days 1-21 of each month (starting Month 1 andcontinuing through Month 12) for approximately 12 months of additionaltreatment.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

NAME SEQUENCE SEQ ID NO Human CD79bRFIARKRGFT VKMHCYMNSA SGNVSWLWKQ EMDENPQQLK 1 precursor;LEKGRMEESQ NESLATLTIQ GIRFEDNGIY FCQQKCNNTS Acc. No.EVYQGCGTEL RVMGFSTLAQ LKQRNTLKDG IIMIQTLLII NP_000617.1;LFIIVPIFLL LDKDDSKAGM EEDHTYEGLD IDQTATYEDI signal VTLRTGEVKW SVGEHPGQE sequence = amino acids 1 to 28 Human matureAR SEDRYRNPKG SACSRIWQSP RFIARKRGFT VKMHCYMNSA 2 CD79b,SGNVSWLWKQ EMDENPQQLK LEKGRMEESQ NESLATLTIQ withoutGIRFEDNGIY FCQQKCNNTS EVYQGCGTEL RVMGFSTLAQ signalLKQRNTLKDG IIMIQTLLII LFIIVPIFLL LDKDDSKAGM sequence;EEDHTYEGLD IDQTATYEDI VTLRTGEVKW SVGEHPGQE amino acids 29 to 229VH of mMAb Gly Pro Glu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys 3anti-CD20 1           5                   10                  5antibody B-Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp Met Asn Trp Val Lys Leu Ly1            20                  25                  30Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Phe Pro Gly Asp        35                  40                  45GLy Asp Thr Asp Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr    50                  55                  60Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Thr Ser Leu Thr65                  70                  75                  80Ser Val Asp Ser Ala Val Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly                85                  90                  95Tyr Trp Leu Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala            100                 105                 110 VL of mMAbAsn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser 4anti-CD20 1               5                   10                  15antibody B-Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Ly1            20                  25                  30Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn        35                  40                  45Leu Val Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr    50                  55                  60Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val65                  70                  75                  80Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly                85                  90                  95Thr Lys Leu Glu Ile Lys Arg             100 GA101 HVR-H1Gly Tyr Ala Phe Ser Tyr 5 1               5 GA101 HVR-H2Phe Pro Gly Asp Gly Asp Thr Asp 6 1               5 GA101 HVR-H3Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr 71               5                   10 GA101 HVR-L1Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr 81               5                   10                  15 GA101 HVR-L2Gln Met Ser Asn Leu Val Ser 9 1               5 GA101 HVR-L3Ala Gln Asn Leu Glu Leu Pro Tyr Thr 10 1               5 GA101 VHGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 111               5                   10                  15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser            20                  25                  30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 GA101 VLAsp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 121               5                   10                  15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser            20                  25                  30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser        35                  40                  45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro    50                  55                  60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65                  70                  75                  80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn                85                  90                  95Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys            100                 105                 110 Arg Thr Val        115 GA101 HeavyGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 13 Chain1               5                   10                  15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser            20                  25                  30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe        115                 120                 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu    130                 135                 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145                 150                 155                 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu                165                 170                 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser            180                 185                 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro        195                 200                 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys    210                 215                 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225                 230                 235                 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                245                 250                 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp            260                 265                 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn        275                 280                 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val    290                 295                 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305                 310                 315                 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys                325                 330                 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr            340                 345                 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr        355                 360                 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu    370                 375                 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385                 390                 395                 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys                405                 410                 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu            420                 425                 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly        435                 440                 445 GA101 LightAsp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 14 Chain1               5                   10                  15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser            20                  25                  30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser        35                  40                  45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro    50                  55                  60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65                  70                  75                  80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn                85                  90                  95Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys            100                 105                 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu        115                 120                 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe    130                 135                 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145                 150                 155                 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser                165                 170                 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu            180                 185                 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser        195                 200                 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210                 215VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 15humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH2)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 16humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH3)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Leu Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 humanized B-QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA 17 Ly1 HeavyPGQGLEWMGR IFPGDGDTDY NGKFKGRVTI TADKSTSTAY ChainMELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSSASTKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSWNSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTYICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDELTKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG humanized B-DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW 18 Ly1 LightYLQKPGQSPQ LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI ChainSRVEAEDVGV YYCAQNLELP YTFGGGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEChuMA79bv28 EVQLVESGGG LVQPGGSLRL SCAASGYTFS SYWIEWVRQA 19 heavy chainPGKGLEWIGE ILPGGGDTNY NEIFKGRATF SADTSKNTAY variableLQMNSLRAED TAVYYCTRRV PIRLDYWGQG TLVTVSS region huMA79bv28DIQLTQSPSS LSASVGDRVT ITCKASQSVD YEGDSFLNWY 20 light chainQQKPGKAPKL LIYAASNLES GVPSRFSGSG SGTDFTLTIS variableSLQPEDFATY YCQQSNEDPL TFGQGTKVEI KR region huMA79bv28 GYTFSSYWIE 21HVR H1 huMA79bv28 GEILPGGGDTNYNEIFKG 22 HVR H2 huMA79bv28 TRRVPIRLDY 23HVR H3 huMA79bv28 KASQSVDYEGDSFLN 24 HVR L1 huMA79bv28 AASNLES 25 HVR L2huMA79bv28 QQSNEDPLT 26 HVR L3 huMA79bv28 EVQLVESGGGLVQPGGSLRLCAAS 27heavy chain (HC) framework region (FR) 1 huMA79bv28 WVRQAPGKGLEWI 28HC FR2 huMA79bv28 RATFSADTSKNTAYLQMNSLRAEDTAVYYC 29 HC FR3 huMA79bv28WGQGTLVTVSS 30 HC FR4 huMA79bv28 DIQLTQSPSSLSASVGDRVTITC 31 light chain(LC) FR1 huMA79bv28 WYQQKPGKAPKLLIY 32 LC FR2 huMA79bv28GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 33 LC FR3 huMA79bv28 FGQGTKVEIKR 34LC FR4 huMA79bv28 DIQLTQSPSS LSASVGDRVT ITCKASQSVD YEGDSFLNWY 35light chain QQKPGKAPKL LIYAASNLES GVPSRFSGSG SGTDFTLTIS (Igκ)SLQPEDFATY YCQQSNEDPL TFGQGTKVEI KRTVAAPSVFIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQSGNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEChuMA79bv28 EVQLVESGGG LVQPGGSLRL SCAASGYTFS SYWIEWVRQA 36 heavy chainPGKGLEWIGE ILPGGGDTNY NEIFKGRATF SADTSKNTAY (IgG1)LQMNSLRAED TAVYYCTRRV PIRLDYWGQG TLVTVSSASTKGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG huMA79bv28EVQLVESGGG LVQPGGSLRL SCAASGYTFS SYWIEWVRQA 37 A118CPGKGLEWIGE ILPGGGDTNY NEIFKGRATF SADTSKNTAY cysteineLQMNSLRAED TAVYYCTRRV PIRLDYWGQG TLVTVSSCST engineeredKGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS heavy chainGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC (IgG1)NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG huMA79bv28DIQLTQSPSS LSASVGDRVT ITCKASQSVD YEGDSFLNWY 38 V205CQQKPGKAPKL LIYAASNLES GVPSRFSGSG SGTDFTLTIS cysteineSLQPEDFATY YCQQSNEDPL TFGQGTKVEI KRTVAAPSVF engineeredIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS light chainGNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV (Igκ) THQGLSSPCT KSFNRGEChuMA79bv28 EVQLVESGGG LVQPGGSLRL SCAASGYTFS SYWIEWVRQA 39 S400CPGKGLEWIGE ILPGGGDTNY NEIFKGRATF SADTSKNTAY cysteineLQMNSLRAED TAVYYCTRRV PIRLDYWGQG TLVTVSSAST engineeredKGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS heavy chainGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC (IgG1)NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDCDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 40humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Val Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH4)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 41humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser(B-HH5)                             20                  25                  30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 42humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH6)            20                  25                  30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 43humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH7)            20                  25                  30Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 44humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser (B-HH8)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 45humanized B- 1               5                   10                  15Ly1 antibodySer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr Ser (B-HH9)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 46humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL8)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90              95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 47humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Tyr Ser (B-HL10)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 48humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL11)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Ala Gly Leu Val Lys Pro Gly Gly 49humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL12)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly 50humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL13)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Lys Lys Pro Gly Gly 51humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL14)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ser 52humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL15)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 53humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL16)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VH ofGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 54humanized B- 1               5                   10                  15Ly1 antibodySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL17)            20                  25                  30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met        35                  40                  45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe    50                  55                  60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65                  70                  75                  80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ser         115 VL ofAsp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 55humanized B- 1               5                   10                  15Ly1 antibodyGlu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser (B-KVI)            20                  25                  30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser        35                  40                  45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro    50                  55                  60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65                  70                  75                  80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn                85                  90                  95Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys            100                 105                 110 Arg Thr Val        115

1-41. (canceled)
 42. A method of treating a B-cell proliferativedisorder in an individual, comprising administering to the individual aneffective amount of (a) an immunoconjugate comprising an anti-CD79bantibody linked to a cytotoxic agent, (b) an anti-CD20 antibody, and (c)a BCL2 inhibitor or a pharmaceutically acceptable salt thereof.
 43. Themethod of claim 42, wherein the anti-CD20 antibody is selected from thegroup consisting of rituximab, obinutuzumab, ofatumumab, ublituximab,and ibritumomab tiuxetan.
 44. The method of claim 42, wherein the BCL2inhibitor is4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamideor a pharmaceutically acceptable salt thereof.
 45. The method of claim42, wherein the immunoconjugate is polatuzumab vedotin.
 46. The methodof claim 42, wherein the anti-CD79b antibody comprises (I) ahypervariable region-H1 (HVR-H1) comprising the amino acid sequence ofSEQ ID NO: 21; (ii) an HVR-H2 comprising the amino acid sequence of SEQID NO: 22; (iii) an HVR-H3 comprising the amino acid sequence of SEQ IDNO: 23; (iv) a hypervariable region-L1 (HVR-L1) comprising the aminoacid sequence of SEQ ID NO: 24; (v) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 25; and (vi) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:
 26. 47. The method of claim 42, wherein theanti-CD79b antibody comprises (i) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 19, and (ii) a lightchain variable domain comprising the amino acid sequence of SEQ ID NO:20.
 48. The method of claim 42, wherein the anti-CD79b antibodycomprises: (i) a heavy chain comprising the amino acid sequence of SEQID NO: 36, and a light chain comprising the amino acid sequence of SEQID NO: 35; (ii) a heavy chain comprising the amino acid sequence of SEQID NO: 37, and a light chain comprising the amino acid sequence of SEQID NO: 35; or (iii) a heavy chain comprising the amino acid sequence ofSEQ ID NO: 36, and a light chain comprising the amino acid sequence ofSEQ ID NO:
 38. 49. The method of claim 42, wherein the anti-CD79bantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 36, and a light chain comprising the amino acid sequence ofSEQ ID NO:
 35. 50. The method of claim 42, wherein the immunoconjugatecomprises the formula Ab-(L-D)p, wherein: (a) Ab is the anti-CD79bantibody; (b) L is a linker; (c) D is the cytotoxic agent, which ismonomethyl auristatin E (MMAE); and (d) p is between 1 and
 8. 51. Themethod of claim 50, wherein the linker comprises (i) a val-citdipeptide; (ii) a Phe-homoLys dipeptide; or (iii) hydrazine.
 52. Themethod of claim 42, wherein the immunoconjugate comprises the formula

wherein Ab is the anti-CD79b antibody, Val is valine, Cit is citrulline,and p is between 1 and
 8. 53. The method of claim 52, wherein theimmunoconjugate is administered to the individual at a dose of about 1.4mg/kg or about 1.8 mg/kg.
 54. The method of claim 52, wherein theimmunoconjugate is administered to the individual at a dose of about 1.8mg/kg.
 55. The method of claim 42, wherein the BCL2 inhibitor isvenetoclax.
 56. The method of claim 55, wherein venetoclax isadministered to the individual at a dose of about 400 mg, about 600 mg,or about 800 mg.
 57. The method of claim 42, wherein the anti-CD20antibody is obinutuzumab.
 58. The method of claim 57, whereinobinutuzumab is administered to the individual at a dose of about 1000mg.
 59. The method of claim 42, wherein i) the immunoconjugate ispolatuzumab vedotin administered to the individual at a dose of about1.4 mg/kg or about 1.8 mg/kg, ii) the anti-CD20 antibody is obinutuzumabadministered to the individual at a dose of about 1000 mg, and iii) theBCL2 inhibitor is venetoclax administered to the individual at a dose ofabout 400 mg, about 600 mg, or about 800 mg.
 60. The method of claim 59,wherein polatuzumab vedotin is administered to the individual at a doseof about 1.8 mg/kg.
 61. The method of claim 59, wherein polatuzumabvedotin, obinutuzumab, and venetoclax are administered during aninduction phase comprising six 21-day cycles.
 62. The method of claim61, wherein (i) polatuzumab vedotin is administered intravenously on Day1 of the first 21-day cycle, venetoclax is administered orally on eachof Days 1-21 of the first 21-day cycle, and obinutuzumab is administeredintravenously on each of Days 1, 8, and 15 of the first 21-day cycle,and (ii) polatuzumab vedotin is administered intravenously on Day 1 ofeach of the second, third, fourth, fifth, and sixth 21-day cycles,venetoclax is administered orally on each of Days 1-21 of each of thesecond, third, fourth, fifth, and sixth 21-day cycles, and obinutuzumabis administered intravenously on Day 1 of each of the second, third,fourth, fifth, and sixth 21-day cycles.
 63. The method of claim 62,wherein (i) venetoclax is administered prior to obinutuzumab andobinutuzumab is administered prior to polatuzumab vedotin on Day 1 ofthe first 21-day cycle, and venetoclax is administered prior toobinutuzumab on Days 8 and 15 of the first 21-day cycle; and (ii)venetoclax is administered prior to obinutuzumab and obinutuzumab isadministered prior to polatuzumab vedotin on Day 1 of each of thesecond, third, fourth, fifth, and sixth 21-day cycles.
 64. The method ofclaim 42, wherein the B-cell proliferative disorder is follicularlymphoma (FL).
 65. The method of claim 62, wherein venetoclax andobinutuzumab are further administered during a maintenance phase afterthe sixth 21-day cycle of the induction phase, wherein venetoclax isadministered orally at a dose of about 400 mg, about 600 mg, or about800 mg once per day during the maintenance phase, and whereinobinutuzumab is administered intravenously at a dose of about 1000 mgonce every two months during the maintenance phase.
 66. The method ofclaim 65, wherein venetoclax is administered for a maximum of 8 monthsduring the maintenance phase.
 67. The method of claim 65, whereinobinutuzumab is administered during the maintenance phase starting onDay 1 of the second month after the sixth 21-day cycle of the inductionphase.
 68. The method of claim 67, wherein obinutuzumab is administeredfor a maximum of 24 months during the maintenance phase.
 69. The methodof claim 68, wherein venetoclax is administered prior to obinutuzumab onDay 1 of each of Month 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24during the maintenance phase.
 70. The method of claim 42, wherein theB-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL).71. The method of claim 62, wherein venetoclax and obinutuzumab arefurther administered during a consolidation phase after the sixth 21-daycycle of the induction phase, wherein venetoclax is administered orallyat a dose of about 400 mg, about 600 mg, or about 800 mg once per dayduring the consolidation phase, and wherein obinutuzumab is administeredintravenously at a dose of about 1000 mg once every two months duringthe consolidation phase.
 72. The method of claim 71, wherein venetoclaxis administered for a maximum of 8 months during the consolidationphase.
 73. The method of claim 71, wherein obinutuzumab is administeredduring the consolidation phase starting on Day 1 of the second monthafter the sixth 21-day cycle of the induction phase.
 74. The method ofclaim 73, wherein obinutuzumab is administered for a maximum of 8 monthsduring the consolidation phase.
 75. The method of claim 74, whereinvenetoclax is administered prior to obinutuzumab on Day 1 of each ofMonth 2, 4, 6, and 8 during the consolidation phase.